Data supporting characterization of CLIC1, CLIC4, CLIC5 and DmCLIC antibodies and localization of CLICs in endoplasmic reticulum of cardiomyocytes

Chloride intracellular channel (CLICs) proteins show 60–70% sequence identity to each other, and exclusively localize to the intracellular organelle membranes and cytosol. In support of our recent publication, “Molecular identity of cardiac mitochondrial chloride intracellular channel proteins” (Ponnalagu et al., 2016) [1], it was important to characterize the specificity of different CLIC paralogs/ortholog (CLIC1, CLIC4, CLIC5 and DmCLIC) antibodies used to decipher their localization in cardiac cells. In addition, localization of CLICs in the other organelles such as endoplasmic reticulum (ER) of cardiomyocytes was established. This article also provides data on the different primers used to show the relative abundance of CLIC paralogs in cardiac tissue and the specificity of the various CLIC antibodies used. We demonstrate that the predominant CLICs in the heart, namely CLIC1, CLIC4 and CLIC5, show differential distribution in endoplasmic reticulum. CLIC1 and CLIC4 both show co-localization to the endoplasmic reticulum whereas CLIC5 does not.

Specificity of CLIC antibodies using specific knock out mice and clic 109 mutant flies.

Experimental features
Confocal microscopy to determine the localization of CLICs in cardiac endoplasmic reticulum, specificity of the antibody used is checked by Western blotting using specific CLIC KO mouse cardiac tissues and Drosophila mutant cardiac tubes.

Data source location
Laboratory animals from Charles Rivers, PA

Data accessibility Data is with this article
Value of the data Our data describing the differential localization of CLICs in cardiac cells provide a possible mechanism for their diverse functional roles in the heart.
Listed antibodies are characterized for their specificity which can be used by other investigators. Table 1 provides the list of primers used to determine the relative abundance of each CLICs in the cardiac tissue [1]. Specificity of the CLIC antibodies was evaluated using the cardiac tissue lysates from the clic1 À / À , clic4 À / À and clic5 À / À mice. Absence of CLIC specific band in the knock out cardiac lysates indicated the specificity of the antibodies used in this study (Fig. 1).
DmCLIC, an ortholog of CLIC in Drosophila melanogaster, localizes to the mitochondria of cardiac tubes [1] as well. Further, specificity of the DmCLIC antibodies was tested in cardiac tubes of CLIC mutant flies, clic 109 (Fig. 3).

Experimental design, materials and methods
The following methods support the results section of the published manuscript describing molecular identity of mitochondrial CLIC proteins.

Real time PCR analysis and quantification of various CLIC transcripts
Hearts from the 2 month old Sprague-Dawley rat were used to prepare total RNA using TRIZOL reagent (Life technologies). This was followed by double digestion with RNase-free DNase for 30 min at 37°C. RNA prepared was further cleaned up with RNeasy mini kit (Qiagen). Purified RNA (2 μg) was reverse-transcribed with Omniscript Reverse Transcription (RT) kit (Qiagen) using oligodT primer. The reverse transcriptase was inactivated by heating at 95°C for 5 min. Real-time qPCR was performed using iQ SYBR Green Supermix (Bio-Rad) in applied biosciences system (iQ cycler, Applied Biosystems), according to MIQE guidelines [5,6]. 1 μl of RT reaction product, and 0. 3 ml of 25 pmole/ml Fig. 1. Specificity of CLIC antibodies using CLIC1, CLIC4 and CLIC5 knock out (KO) mice heart lysates. (I) 50 µg of heart lysates from WT, clic1 À / À , clic4 À / À and clic5 À / À mouse were electrophoresed on 4-20% (w/v) SDS-PAGE, transferred onto nitrocellulose membrane and probed with anti-CLIC1, anti-CLIC4 and anti-CLIC5 antibodies. Absence of CLIC1, CLIC4 and CLIC5 specific bands in KO organ samples confirms the specificity of the antibody. Corresponding Ponceau S stained nitrocellulose membrane is shown as a loading control.
primer pairs (Table 1) were added in a 20-μL reaction. Primer pairs were designed to flank an intron to control contamination from genomic DNA. The amplification conditions comprised an initial denaturation step at 95°C for 5 min, and 40 cycles of 95°C for 45 s, 60°C for 45 s, and 72°C for 45 s. Mock cDNA (no reverse transcriptase) as well as primers to amplify GAPDH were used as a control to assess the quality of the cDNA and genomic DNA contamination. GAPDH will be used to normalize the expression of different CLICs. All samples were run in triplicates. Threshold cycle values (Cq) were measured at a fluorescence of 100 a.u. Efficiency of the primers was calculated using the slope of the standard curve plot (threshold cycle vs. log of various DNA concentration) defined as (10-1/slope-1) Â 100. Clear single peaks at their melting temperature and a clear band at the expected size in agarose gels confirmed the amplification of specific products.

Super resolution microscopy
STED images were acquired with a custom-made STED nano-scope using an oil immersion objective (HCX PL APO 100 Â /1.40-0.70 OIL CS, Leica Germany) as described earlier [8]. A 635 nm pulsed diode laser (LDH-D-C-635, PicoQuant GmbH) was used for excitation. A tunable Ti:sapphire laser (Mai Tai HP, Spectra Physics) set at 780 nm was used to deliver pulses for STED depletion. Fluorescence emission from ATTO 647N-labeled secondary antibodies was collected through a Semrock BrightLine FF01-692/40-25 nm band pass filter in front of a photomultiplier (H7422PA-40, Hamamatsu Photonics K.K.). Images (955 Â 960 pixels) were acquired with a 16 kHz line frequency (resonant mirror of 8 kHz) and summed 256 times. Pixel size was $ 9.575 nm Â 9.575 nm. For comparison between conventional confocal images and STED images, all imaging parameters were kept identical except for the number of summations which was 64 when recording confocal images. Confocal images were acquired first for the same field prior to STED imaging. For analysis, STED images were filtered by mean subtraction with a filter window width and height of 32 and threshold was set to 1 [9].

Sub-cellular fractionation of mitochondria
Mitochondria was sub-fractionated as described earlier [10]. Briefly, hearts from 2 months old rats were excised, washed with PBS and then homogenized in mito-isolation buffer (mmole/L, 3 HEPES-KOH, 210 mannitol, 70 sucrose, 0.2 EGTA, complete mini EDTA-free protease inhibitor cocktail) followed by centrifugation at 2500g for 5 min at 4°C. The supernatant was spun again at 12,000g for 10 min for separating mitochondria at 4°C. The pellet enriched with mitochondria was then resuspended in mito-isolation buffer containing 2.5 mg/mL of digitonin and then vortexed for 15 min. The suspension was then centrifuged again at 12,000g for 10 min at 4°C. The supernatant containing outer mitochondrial membrane and inter-membrane space was transferred to another tube. The pellet was resuspended in 500 μl of mito-isolation buffer containing 2.5 mg/mL of digitonin and then sonicated briefly in ice-cold water sonicator. This was followed by centrifugation at 100,000g for 30 min at 4°C. After centrifugation the pellet containing inner mitochondrial membrane was stored for further analysis in Western blot.