Anti vdac1 porin

Immediately following RSV and/or LSS applications, HUVECs were harvested for protein analysis. Aliquots from cell lysates were separated by SDS-PAGE on 10% gels and transferred onto PVDF membranes, which were blocked with 5% non-fat dry milk dissolved in Tris-buffered saline and then incubated overnight with primary antibodies at 4°C. Immunoreactive proteins were detected by chemiluminescence with Thermo Scientific SuperSignal (Pierce Biotechnology, IL). The primary antibodies used included anti-SIRT1 (Cell Signaling Technology, Danvers, MA), anti-PGC-1α (NOVUS, Littleton, CO), anti-TFAM (NOVUS, Littleton, CO), anti-VDAC1/Porin (ABcam, Cambridge, MA), and anti-α-tubulin (Sigma, St Louis, MO).

Immunoblotting was performed as described previously^{19 (link)}. Briefly, differentiated myotubes were lysed in RIPA buffer (10 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, pH 7.5) with cell scraper. Following centrifugation (16,000 g for 15 min at 4°C), supernatants were collected for Bradford assay to quantify total protein concentrations. Following SDS-PAGE, samples were transferred to the PVDF membrane. Subsequently, the membranes were incubated in TBST containing 5% nonfat dry milk for 30 min prior to overnight incubation with respective primary antibodies (rabbit polyclonal anti-VDAC1/porin [Abcam] and mouse monoclonal α-tubulin [Sigma-Aldrich]). After washing twice in TBST, the membranes were further incubated with HRP-conjugated secondary antibodies for 40 min, followed by washing thrice with TBST. Then, membranes were subjected to standard enhanced chemiluminescence (Thermo Fisher Scientific) method for visualization. The resulting band intensities were quantified by ImageJ software (NIH).

Cells were lysed in RIPA buffer (150 mM NaCl, 1.0% Nonidet NP-40, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) supplemented with protease and phosphatase inhbibitors for 30 min with shaking on ice. Protein content was determined by the BCA assay (Pierce, Rockford, IL, USA). Samples were boiled for 5 min in reducing loading buffer before separation on SDS-PAGE gels. Wet blotting was used to transfer the separated samples to nitrocellulose membranes (Whatman-GE, Little Chalfont, UK). Immunoblotting was done in TBS/tween supplemented with 5% non-fat dried milk overnight at 4 ºC. Following antibodies were used: anti-H-Ras (Santa Cruz, Dallas, TX, USA, sc-520), anti-actin HRP labeled (Cell Signaling, Danvers, MA, USA, 5125), unlabeled actin (Millipore, Darmstadt, Germany, MAB1501, used for Figure 2b), anti-cleaved caspase 3 (Cell Signaling, 9664), anti-catalase (Abcam, Cambridge, UK, Ab1877), Anti-Vdac1/porin (Abcam, ab15895), anti-SDHA (Abcam, ab14715), anti-ATPase alpha subunit (Abcam, ab14748). Rabbit polyclonal antibody to GPD2 was custom prepared.^{45 (link)} HRP-conjugated secondary antibodies were used in TBS/tween with 5% non-fat dried milk for 1 h at room temperature. WB signals were quantified using the Aida 3.21 Image Analyzer software (Raytest, Straubenhardt, Germany).