Mice.
MC-deficient mice (Wsh/Wsh; ‘Sash’) were purchased from Jackson Laboratories and bred in-house at Duke-NUS Medical School vivarium, Singapore. Germ-free mice on the C57BL/6 background were purchased from either Biological Resource Center (BRC), Agency for Science, Technology, and Research (A*STAR), Singapore)/In Vivos (Singapore), and maintained in a germ-free facility. Nlrp3−/− were procured from the University of Lausanne (Switzerland) and Asc−/− and Caspase-1−/− from Genetech (USA) and were maintained at BRC (A*STAR), Singapore, under specific pathogen-free conditions. All animal experiments were performed in compliance with the guidelines from the national care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (IACUC), SingHealth (Protocol # 2015/SHS/1121), and BRC, A*STAR (Protocol #161113), Singapore.
Methods Details
Cell culture.
To generate BMMCs for both in vitro experiments and in vivo reconstitution studies, bone marrow was flushed from mouse femurs and cultured in RPMI medium containing 10% FBS (Gibco BRL), 100 U/ml penicillin, 0.1 mg/ml streptomycin, Hepes, sodium pyruvate, the non-essential amino acid (Invitrogen, Life Technologies, Singapore), and recombinant murine IL-3 (3 ng/ml) (Immuno Tools, Cat # 12340033) and SCF (3 ng/ml, Immuno Tools, Cat # 12343323). Rat basophilic leukemia-2H3 mast cells (RBL-2H3) were cultured in α-MEM (Invitrogen, Life Technologies, Singapore) supplemented with 10% FBS, 100 U/ml penicillin/ 100 µg/ml streptomycin (GIBCO/Thermo Fisher, Cat # MT30002CI) in a 5% CO2 incubator at 37°C. HEK293T cells were cultured in α-DMEM (Invitrogen, Life Technologies, Singapore) supplemented with 10% FBS (Invitrogen, Life Technologies, Singapore), 100 U/ml penicillin, and 100 µg/ml streptomycin in a 5% CO2 incubator at 37°C. LAD2 human mast cells were propagated in a complete StemPro-34 medium (Thermo Fisher, Cat # 10639011) supplemented with 100 ng/mL rhSCF (R&D Systems, Cat # 255-SC-010/CF).
In vivo mouse studies.
Reconstitution of Sash mice was performed by injecting 1 x 107 BMMCs intravenously (i.v.) followed by a 12–16 weeks period before use in experiments to allow full engraftment, as previously described 36. For passive systemic anaphylaxis studies, mice were intraperitoneal (i.p.) injected with anti-TNP IgE (5 µg/mouse, BD Biosciences, Cat # 557080) 2–4 days before the challenge i.v. with TNP-OVA (250 µg/mouse, Biosearch Technologies, Cat # T-5051). Body temperature was recorded using an intrarectal probe (Physitemp, model BAT-12) at the times indicated. The in vivo CY-09 (Sigma Aldrich, Cat # SML2465) selective inhibitory activity of MCs NLRP3/ASC was confirmed by the passive systemic anaphylaxis using MC-deficient mice repleted with BMMCs from WT, Asc−/−, or Nlrp3−/− mice sensitized with anti-TNP IgE and pretreated or not with CY-09 (100 µg /mouse i.p.) 1 h before the challenge with TNP-OVA. To investigate, the contribution of IL-1β in passive systemic anaphylaxis after the anti-TNP IgE sensitization (5 µg/mouse), mice were injected i.p. with TNP-OVA (150 µg/mouse) alone or with recombinant murine IL-1β (5 or 50 ng/mouse; Immuno Tools, Cat # 12340012) or purified anti-mouse IL-1β antibody (150 µg/mouse, Clone B122, Biolegend, Cat # 515801). In another set of experiments, LPS (Sigma Aldrich, Cat # L2880) pretreatment i.p. (0.3 µg/mouse) was done or not, 90 min before the i.p. OVA-TNP challenge in combination with anti-mouse IL-1β antibody (150 µg/mouse). Purified Armenian Hamster IgG antibody (150 mg/mouse, Clone HTK888, Biolegend, Cat # 400911) was used as an isotype control.
Mast cell degranulation assay.
WT, Nlrp3−/−, Asc−/− BMMCs (2 x 105/well) were sensitized overnight at 37 oC with a 0.5 µg/ml anti-DNP IgE antibody in 96-wells plate and were stimulated for 30 min with various concentration of DNP-BSA (Sigma Aldrich). Where indicated, the cells were pre-treated for 20–30 min with Ciliobrevin D (Merck Millipore, Cat # 250401), CY-09 (Cat # SML2465, or CAY-10736 (Cayman, Cat # Cay26754-1), or Oridonin (Sigma Aldrich, Cat # O9639) or PF-431396 (Sigma Aldrich, PZ0185), respectively. Samples were centrifuged, and the supernatant was collected to measure the released β-Hexosaminidase (Sigma Aldrich, Cat # N9376 and histamine (Enzo Life Sciences, Cat # ENZ-KIT140-001). To evaluate the total cell count of β-Hexosaminidase, cells were lysed in 0.5% Triton-X100 in PBS buffer. For degranulation assay, 100 µl of supernatants or cell lysates were incubated with 50 µl p-nitrophenyl-N-acetyl-β-D-glucosaminide (1.3 mg/ml in 0.1 M sodium citrate, pH 4.5), and incubated for 1 h at 37°C. The reaction was stopped by the addition of 150 µl 0.1 M carbonate buffer pH 10 and measured at OD405 nm with a microplate reader (Tecan). The percentage of degranulation was calculated by dividing absorbance in the supernatant by the sum of absorbance in the supernatant and cell lysate.
Knockdown of Nlrp3, Asc, and Caspase-1 in RBL-2H3 mast cells.
RBL-2H3 cells (500,000 cells/well) were transfected with 100 nM of each siRNA procured from Dharmacon containing control (Cat # D-001810-01) or Asc (Cat # L-097663-02) or Nlrp3 (Cat # L-084509-02) or Caspase-1 (Cat # AM16708) individually or in combination 100 nM of each siRNA (Nlrp3 + Asc) in 24-well dishes using X-Fect (Clontech, Cat # 631317) according to the manufacturer’s instructions(Dharmacon). After 48 h, the cells were stimulated with IgE/Ag (0.5 µg/ml)/Ag (1 µg/ml) for 45 min at 37 oC, and MC degranulation was monitored as described in the MC degranulation assay section.
Ectopically expression of NLRP3 and ASC in Nlrp3−/− or Asc−/− BMMCs.
BMMCs obtained from either Nlrp3−/− or Asc−/− (2 x 107) or WT were nucleofected with 5 µg of each expression plasmids expressing mouse Flag-tagged NLRP3 (Addgene, Cat # 75127) or ASC (Addgene, Cat # 75134) or empty plasmid using P3 Primary Cell 4D-Nucleofector X-kit (Lonza). After 36 h, the MCs were stimulated with (0.5 µg/ml)/Ag (1 µg/ml) stimulation. Degranulation was measured by the release of β-hexosaminidase levels in the culture medium as described under degranulation assay.
Calcium measurement assay.
The Ca2+ influx was determined in BMMCs from WT, Nlrp3−/−, and Asc−/− BMMCs loaded with Fluo-4 NW probe (Thermo Scientific, Cat # F36206) following the manufacturer’s instructions. Cells were stimulated with IgE/Ag, or thapsigargin (100 nM) (Sigma Aldrich, Cat # T9033). Fluorescence intensities were measured using a Tecan spectrophotometer.
Measurements of MC-derived mediators.
BMMCs were seeded after anti-DNP IgE antibody sensitization at the concentration of 3 x 105 /well/200 µL and stimulated with DNP-BSA (0.5 µg/ml), or primed with LPS (250 ng/ml for 3–4 h) and stimulated with nigericin (Sigma Aldrich). The cell culture supernatants were harvested, as indicated in the Figure legend, and subjected to ELISA quantification for TNF-α, IL-6, IL-1β (e-Bioscience, Cat # BMS607-3, 29-8061-65, BMS6002), histamine (Enzo Life Sciences, Cat # ENZ-KIT140-001), and PGD2 (Cayman Chemical, Cat # 512031). IL-1β was also quantified in the supernatant of WT and Nlrp3−/− BMMCs (2 x 106/well/ml) with or without TNP-OVA (0.5 µg/ml) treatment for 30 min after anti-TNP IgE (0.5 µg/ml) and LPS (250 ng/ml) primed for 4 h.
Flow cytometry.
For CD63 externalization, MCs were fixed (4% PFA) for 20 minutes at room temperature, then rinsed three times in PBS for 5 min and incubated with α-CD45, α-FcεRI, α-CD117 and α-CD63 (Biolegend, 1:100) for 30 minutes at 4 ⁰C. For the detection of MC maturation, cells were resuspended in PBS w/o Ca2+ and Mg2+ containing 2 mM EDTA, 2% FBS and stained with CD45, FcεRI and CD117 (Biolegend), followed by fixation, permeabilization with BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD Bioscience) and intracellularly stained for MC granules using the granule heparin-specific probe avidin-FITC conjugated (eBioscience) for twenty minutes. MCs granularity was confirmed to be > 95% pure by toluidine blue (Sigma-Aldrich, Singapore).
Immunofluorescent microscopy.
Cells were fixed in 4% paraformaldehyde for 20 min, then rinsed three times in PBS for 5 min and incubated in blocking buffer (0.1% Saponin (Sigma Aldrich, Cat # 47036) in 1% BSA-PBS) for 30 min at room temperature. The cells were then incubated with α-mouse CD63 (MBL, Rat IgG 1:100, Cat # D263-3) or α-mouse ASC (EMD Millipore, mouse IgG, 1:1000, Cat # 04-147) or α-mouse NLRP3 (Novus Biological, rabbit IgG 1: 50, Cat # NBP2-12446) or α-mouse α-tubulin (Sigma Aldrich, mouse IgG, 1:250, Cat # T5168) or α-mouse α-dynein (Sigma Aldrich, mouse IgG, 1: 250, Cat # MAB1618) or α-mouse IL-1β (Abcam, rabbit IgG, 1:100, Cat # ab9722) diluted in blocking buffer for 3 h. Cells were then washed twice in blocking buffer for 5 min and incubated overnight at 4o C in blocking buffer. The next day the cells were washed once in blocking buffer for 5 min and incubated with the following secondary antibodies: goat α-rat IgG AF488 (Thermo Scientific, Cat # A-11006) for CD63, chicken anti-rabbit IgG AF568 (Abcam, ab175470) for NLRP3 and IL-1β, donkey anti-mouse IgG AF647 (Abcam, Cat # ab6706) for ASC, donkey anti-mouse IgG AF568 (Abcam, Cat # ab175472) for dynein and chicken anti-mouse IgG AF568 (Abcam) for α-tubulin detection, for 1 h at room temperature, diluted 1: 400 in blocking buffer. Cells were then washed 4–5 times, incubated overnight at 4oC in blocking buffer, and mounted. All the images were acquired on LSM 710 Carl Zeiss microscope using Plan-Apochromat 63x/1.40 oil DIC objective. Images were acquired at 16-bit depth at a resolution of 1024 x 1024 pixels. The α-tubulin signals were measured in each cell with a radius of 12–16 µm using ZEN Imaging Software (Zeiss). Background fluorescence using a circle of the corresponding size was subtracted from each measurement.
Endogenous and ectopic protein interaction assays and Western blotting.
For analyzing endogenous protein interactions, IgE-sensitized WT, Nlrp3−/− or Asc−/− BMMCs (6 x 107 cells) were pretreated with either CAY-10736 (10 µM) or oridonin (10 µM) or BAPTA-AM (10 µM, Sigma Aldrich, Cat # A1076) or PF-431396 (10 µM) for 30 min following Antigen, DNP-BSA (1 µg/ml) or LPS/nigericin stimulation at different time intervals. The cells were washed in ice-cold PBS and lysed in 1X IP lysis buffer (Cell Signalling, Cat # 9803) containing 1mM phenylmethylsulfonyl fluoride (PMSF) (Sigma Aldrich, Cat # P7626-1G), 1 mM sodium orthovanadate (Sigma Aldrich, Cat # S6508), 1X protease/phosphatase inhibitors (Roche). Total cell lysates (1 mg protein equivalent) were incubated with G-plus agarose beads (25 µl, Santa Cruz, Cat # sc-2002) for 2 h. The pre-cleared cell lysates were immunoprecipitated with 1 µg of each pull-down antibody, CD63 (Santa Cruz, Cat # sc-5275), NLRP3 (Novus Biologicals), ASC (Santa Cruz, sc-514414), and or NEK7 (Novus Biologicals), Pyk2 (Cell Signaling, Cat # 3480), and tumbled overnight at 4°C. The following day, immunocomplexes were precipitated with 30 µl of fresh G-plus agarose beads. After 6–8 h, immune complex precipitates were then extensively washed three times with ice-cold lysis buffer. These precipitates or total cell lysates (TCL) were then subjected o 12 % SDS-PAGE and immunoblotted with corresponding antibodies. Anti-IgG of either rabbit (Abcam, Cat # ab37415) or mouse (Abcam, Cat # ab37355) origin was used as an isotype control. The images were captured using touch screen Gel Doc (Bio-Rad).
For analyzing overexpression protein interaction studies, GFP-CD63 (10 µg, SinoBiological, Cat # MG50557-ANG), Flag-NLRP3/-PYD/-NBD/-LRR (10 µg each, Addgene, Cat # 75137, 75140, 75141), and Flag-ASC/-PYD/-CARD truncate (10 µg each, Addgene, 75134); or GFP-CD63 full length/or its truncates, GFP-tagged TM1, TM2, TM3, and TM4, or 1 and 5 µg of Flag-tagged NLRP3 or Myc-tagged ASC with a constant amount of GFP-CD63-TM3 (5 µg) were transfected into HEK293T cells (700,000 cells/well) using JetPrime transfection reagent (Polyplus). After 36 h, cell lysates were lysed and incubated with 1 µg of Flag (Cell Signalling, Cat # 8146) or GFP (Santa Cruz, Cat # sc-9996) or Myc (Cell Signalling, Cat # 5605) antibodies. The immune complexes were washed thrice in 1X IP Wash buffer, and eluted with 4X-SDS loading dye, and separated onto 4–20 % SDS–PAGE (BioRad) followed by Western blot analysis with the Flag or Myc or GFP antibodies respectively.
For analyzing total/phosphorylation status of respective signaling molecules, the antibodies were procured from Cell Signalling (used in 1:1000 dilution), Syk (Cat # 13198), pSyk (Tyr525/526) Cat # 2710), PLCγ (Cat # 2822), pPLCγ (Cat # 14008), ERK1/2 (Cat # 4695), pERK1/2 (Cat # 4377), p38 (Cat # 8690), p-p38 (Cat # 4511), JNK (Cat # 9252), pJNK (Cat # 9255), Pyk2 (Cat # 3292S), pPyk2 (Cat # 3291), NEK7 (Novus Biological, Cat # NBP-31110) or pASC (ECM, AP5631, Cat # AP5631) respectively. WT, or Nlrp3−/− or Asc−/− BMMCs pretreatment with or without PF-431396 following IgE/Ag stimulation at different time intervals, the cell lysates were lysed in Western buffer (150 mM NaCl, 20 mM Tris-HCl, pH7.4, 1 mM EDTA plus freshly prepared protease inhibitors), incubated on ice for 30 min. The samples were centrifuged at 14,000x g for 10 min. The lysates were quantitated using Bradford’s assay. An equal amount of protein lysates were mixed with 2X SDS-loading dye and boiled at 100 0C for 10 min. The samples were fractionated onto 4–20% SDS-PAGE gels (Bio-Rad) and subjected to Western blot analysis.
Size exclusion chromatography.
WT BMMCs (5 x 107/ml) were stimulated with IgE/Ag for 5 min or LPS (12 h)/nigericin (Sigma Aldrich) for 15 min, respectively. The cells were homogenized with the protein extraction buffer A (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM Na-EDTA, 1 mM EGTA), and freshly prepared protease inhibitor cocktail (Roche). Samples were then centrifuged at 18,000x g for 10 min at 4°C. Sephacryl 100 HR size-exclusion chromatography (SEC) matrix (Sigma Aldrich, Cat # S100HR) was pre-equilibrated in buffer B (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% octyl glucoside, plus protease inhibitor cocktail. Protein standards (IgM, 970 kDa; Thyroglobulin, 669 kDa; β-amylase, 200 kDa; BSA, 66 kDa, cytochrome C, 12 kDa) were procured from Sigma Aldrich (Cat # MWGF1000) were run on a column, and the fractions were monitored at A260 nm. The column was washed and the clear supernatant (100 µl) obtained from control or IgE/Ag or LPS/nigericin (Sigma Aldrich, Cat # N7143) treatments was loaded onto the SEC column separately. Fractions (100 µl) were collected, starting at the void volume time. Equimolar amount (45 µl) of the fractions were mixed with 4X SDS-loading dye, boiled at 100°C for 10 min, and then fractionated onto 4–20% SDS-PAGE gels followed by Western blot analysis with NLRP3, ASC, Caspase-1, and CD63 antibodies, respectively.
Oligomerization assay.
WT BMMCs (1 x 107/ml) were either sensitized with IgE (0.5 µg/ml) or primed with LPS (50 ng/ml) for 12 h. The medium was then replaced, and cells stimulated with nigericin (10 µM) or Ag (OVA-DNP, 1 µg/ml) for 15 and 5 min, respectively. The supernatants were removed, cells were rinsed in ice-cold PBS and then lysed by NP-40 for 30 min. Lysates were centrifuged at 330x g for 10 min at 4°C. The pellets were washed gently twice in 1-ml of ice-cold 1X PBS and resuspended in 500 µl of PBS. Disuccinimydylsuberate (DSS, 2 mM, Sigma Aldrich, Cat # 225827) was added to the resuspended pellets and incubated at room temperature for 30 min with gentle rotation. Samples were then centrifuged at 330x g for 10 min at 4°C. The cross-linked pellets were resuspended in 30 µl sample buffer, boiled, and analyzed by Western blot using antibodies against NLRP3, ASC, and CD63 respectively.
Microtubule’s polymerization assay.
BMMCs (4 x 107) obtained from WT, Nlrp3−/−, Asc−/− and Caspase1/11−/− mice were stimulated with IgE/Ag for 5 min and subjected to polymerization assay as described previously 50. Briefly, cells were lysed in buffer A (250 mM sucrose, 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 1 mM DTT, 0.1% NP-40) and freshly prepared protease inhibitors. The lysates were centrifuged for 10 min at 700x g, and the supernatants were centrifuged for 10 min at 12,500x g. The resultant supernatants were used as cytosolic fraction (soluble) and the pellets were resuspended in 10 mM HEPES buffer (pH 7.4) containing 0.5% NP-40, 50 mM KCl, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA and protease inhibitors. Then, the resuspended pellets were recentrifuged and the supernatants were used for the membrane-enriched fraction (insoluble). Subcellular fractionated lysates were lysed in 4X SDS-loading dye, boiled, and briefly centrifuged. An equal amount of samples were loaded onto 4–20% (Bio-Rad) SDS-PAGE gels, and analyzed by Western. Insoluble samples were separately stained with Coomassie brilliant blue (CBB) served as a loading control.
Subcellular organelle fractionation.
RBL-2H3 cells (1 x 107) were silenced with 100 nM of each siRNA specific for Nlrp3 or Asc or control using X-Fect transfection reagent. After 24 h, the respective wells were transfected with GFP-tagged CD63 (5 µg) plasmid for another 30 h followed by IgE/Ag stimulation for 5 min. The cell lysates were subjected to organelle fractionation as described previously50 with some modifications. Briefly, cells were lysed in buffer A containing 250 mM sucrose, 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 1 mM DTT, 0.1% NP-40 and protease inhibitors. The lysates were centrifuged for 10 min at 700x g, and the supernatants obtained were further centrifuged for 10 min at 12,500x g. The resultant supernatants were used as a cytosolic fraction, and the pellets were resuspended in 10 mM HEPES buffer (pH 7.4) containing 0.5% NP-40, 50 mM KCl, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, and protease inhibitors. Then, the resuspended pellets were recentrifuged, and the supernatants were used for the membrane-enriched fraction. Subcellular fractionated proteins were lysed in buffer containing 2% SDS and boiled with 2X reducing sample buffer and analyzed for GFP, membrane marker- pan-Cadherin (Cell Signalling, Cat # 4068) expression. The blots were also evaluated for ASC and NLRP3 antibodies to determine the knockdown efficiency.
Knockdown or pharmacological inhibition of dynein in RBL-2H3 MCs.
RBL-2H3 cells were silenced for Dynein (200 nM) or control siRNA (200 nM) using an X-Fect transfection reagent. Four different siRNA duplexes were designed against different regions of the DHC sequence using Dharmacon's custom SMARTpool siRNA service (rat cytoplasmic DHC1, 5’-gggaggaggttatgtttaa, 5’-ggtgacagctttcgaatga, 5’-ccaaatacctacattactt, 5’-gctcaaacatgacagaatt). The non-targeted duplex III (Dharmacon, Cat # D-001810-0X) was used as control. After 36 h, cells were primed with IgE (0.5 µg/ml) overnight, and subsequently pretrerated with or without Ciliobrevin D (10 µΜ), for 30 min, and then stimulated with OVA-DNP (1 µg/ml) antigen for 5 min. The lysates were pulled down with the CD63 antibody and subjected to Western blot analysis using Tubulin, CD63, and GAPDH (Cell Signaling, Cat # 9482) antibodies, respectively. Total cell lysates (TCL) were loaded as a control IP input and the silencing of dynein (Santa Cruz, Cat # sc-13524).
Knockdown of Nlrp3 and Asc in human LAD2 MCs.
LAD2 MCs were maintained in complete StemPro-34 medium supplemented with 100 ng/mL rhSCF (R & D Systems, Cat # 255-SC-010/CF). LAD2 (5 × 107 cells) were nucleofected with 200 nM of each siRNA targeting Nlrp3, Asc, or scrambled/control using P3 Primary Cell 4D-Nucleofector X-kit (Lonza, Cat # V4XP-3032). siRNA were customs synthesized from IDT Technologies (Singapore). After 36 h, the LAD2 cells were sensitized with biotinylated human IgE (100 ng/ml, BioLegend, Cat # 325503) overnight, and the cells were washed with HEPES (10 mM HEPES, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.4 mM Na2HPO4, 5.6 mM glucose, 1.8 mM CaCl2, 1.3 mM MgSO4) containing 0.02% BSA, and/or pre-treated with CY-09 (10 µM) for 15 min following Antigen (Streptavidin, 100 ng/ml, Cat # S4762) stimulation for 30 min at 37 oC. Degranulation was measured by the release of β-hexosaminidase levels in the culture medium as described under degranulation assay.
Phenol-chloroform precipitation of cell-free culture supernatants.
For detecting IL-1β (pro-or mature) forms in the extracellular medium, cell culture supernatants (500–1000 µl) from WT, or Nlrp3−/− BMMCs from various stimulations were subjected to phenol: chloroform precipitations. Briefly, the supernatant was precipitated by adding 1/4 volume of chloroform and 1 volume of methanol. The mixture was centrifuged for 5 min at 12,000 rpm. The top layer containing methanol was aspirated out and replaced with a fresh 1 volume of methanol. The mixture was again centrifuged for 5 min at 12,000 rpm. The pellet obtained was air-dried (10–15 min) and dissolved in 1X Western buffer. Alternatively, WT BMMCs cells stimulated with LPS + IgE/Ag were centrifuged at 200x g for 2 min. The supernatant obtained was further centrifuged at 14000 g for 10 min. The pellet fraction so obtained is granule remnants plus and the supernatant is granule remnant free fraction. The granule remnant plus fraction was further processed using gentle sonication (to break down mast cell granules membrane). These two fractions were subjected to precipitation as described above. The precipitated sample (typically 50 µl) was analyzed by 4–20% SDS-PAGE (Bio-Rad) and immunoblotting (pro-IL1β (Abcam, Cat # ab8722) and Chymase (Abcam, Cat # ab233103) antibodies).
Densitometric analysis.
The chemiluminescent blots were imaged first with the ChemiDoc (BioRad). The Band Analysis tool of ImageLab software version 4.1 (BioRad) was used to select and determine the band's intensity in all blots.
Statistical analysis.
Statistical analyses were performed using GraphPad Prism version 9 (GraphPad Software, San Diego, CA). Data are represented as the mean ± standard error (SEM). One-way ANOVA was used for comparisons of > 2 groups, followed by Dunnett's multiple comparisons test. A two-tailed, unpaired Student’s t-test was used to compare 2 groups of data. P-values < 0.05 were considered significant.