Kaposi’s sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies
Fig 5
Dcp1a protein levels are decreased by KapB expression and Torin treatment.
A: HUVECs were treated with DMSO or Torin (250 nM) for 4 h prior to harvest. Samples were lysed in 2X Laemmli buffer and resolved by SDS-PAGE before immunoblotting for Xrn1, EDC4/Hedls, Dcp1a, and DDX6. Samples were quantified by normalizing the PB resident protein levels to the total protein in each lane and then the DMSO control using ImageLab (BioRad). Results were plotted in GraphPad and a one-way ANOVA was performed ±SEM; n = 3, ** = P<0.01. B: HUVECs were transduced with recombinant lentiviruses expressing either KapB or an empty vector control and selected with blasticidin (5 μg/mL). Cells were treated with DMSO or Bafilomycin A1 (BafA1, 10 nM) for 4 h prior to harvest in 2X Laemmli buffer. Samples were resolved by SDS-PAGE and immunoblot was performed for Dcp1a or p62 (autophagy marker). Samples were quantified by normalizing Dcp1a protein levels to the total protein in each lane using Image Lab (BioRad) and then to the vector DMSO control. Representative blot is from the same membrane, hashed line indicates skipped lanes. Results were plotted in GraphPad and a 2-way ANOVA was performed, ±SEM; n = 3, *** = P<0.001. C: HUVECs were transduced with recombinant lentiviruses expressing either KapB or an empty vector control and selected with blasticidin (5 μg/mL). Samples were harvested in 2X Laemmli buffer, resolved by SDS-PAGE and immunoblot was performed for Xrn1, Hedls/EDC4, or DDX6. Samples were quantified by normalizing PB protein levels to the total protein in each lane using Image Lab (BioRad) and then to the vector control. Results were plotted in GraphPad. D: HUVECs were sequentially transduced: first with recombinant lentiviruses expressing either shRNAs targeting Atg5 (shAtg5) or a non-targeting control (NS) and selected with puromycin (1 μg/mL), and second with either KapB or an empty vector control and selected with blasticidin (5 μg/mL). Samples were harvested in 2X Laemmli buffer and resolved by SDS-PAGE. Immunoblot was performed for Dcp1a, Atg5, and KapB. Samples were quantified by normalizing Dcp1a protein levels to the total protein in each lane using Image Lab (BioRad) and then to the vector NS control. Results were plotted in GraphPad and a 2-way ANOVA was performed, ±SEM; n = 3, * = P<0.05, **P = <0.01.