Anti mortalin

Control and treated cells were given a washing with chilled 1X PBS followed by fixation with acetone and methanol (1:1) and permeabilization with 0.3% Triton- X100 in 1X PBS. Cells were then blocked with 2% BSA and incubated with primary mouse monoclonal antibody anti-α-Tubulin (1:500), anti-NF-κB (1:500), anti-MAP-2 (1:250), anti-NF200 (1:500), anti-GAP 43 (1:250), anti-HSP70 (1:500), anti-Mortalin (1:500), anti-Bcl-xL (1:200), anti-Cyclin D1(1:250), anti-NCAM (1:250), rabbit monoclonal anti-AP-1(1:250) (all from Sigma-Aldrich) and mouse monoclonal anti-PCNA (1:250), mouse polyclonal anti-PSA-NCAM (1:250) (from Millipore, MA, USA) for 24 h in humid chamber at 4 °C. No permeabilization was carried out for PSA-NCAM immunostaining. After primary antibody incubation, three washings were given with 0.1% PBST and incubated with secondary antibody (goat anti-mouse/ rabbit IgG/ IgM Alexa Fluor 488/543) for 2 h at RT. Cells were stained with nuclear staining dye DAPI (Sigma-Aldrich) for 15 min, washed with 0.1% PBST and mounted with antifading agent Fluoromount (Sigma-Aldrich). Images were captured with Nikon AIR Confocal Laser Scanning Microscope and analyzed with NIS elements analysis software version 4.11.00 (Nikon Co., Tokyo, Japan). Each experiment was carried out in triplicate.

Cells were washed twice in 1× PBS, pH 7.4, and lysed in buffer A (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 1.5 mmol/L MgCl₂ and 0.5% NP-40). Mitochondria were lysed directly in 1× SDS loading buffer. Protein lysates (50 μg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, incubated for 1 h with 5% milk TBS-T and overnight with primary antibodies in 5% BSA at 4 °C or for 1–2 h at room temperature. Antibodies included anti-PNPASE (1:5,000) (Chen et al., 2006 (link)), anti-TIM23 (1:1000) (Abgent), anti-Mortalin (1:10,000) (Sigma-Aldrich), anti-Creb (1:1000) (Abcam), anti-ActB (1:2,000) (ABClonal), anti-Acetylated lysine (1:1000) (Cell Signaling Technology), anti-TERT (1:1000) (Abcam), anti-p-AMPK (Thr172) (1:1000) (Cell Signaling Technology), anti-SIRT1 (1:500) (Santa Cruz Biotechnology), anti-SIRT6 (1:1000) (Cell Signaling Technology), anti-SIRT3 (Cell Signaling Technology), anti-p-NF-kB(Ser536) (1:1000) (Cell Signaling Technology), anti-p16 (1:1000) (Bioworld) and anti-β-Tubulin (1:2000) (Abcam).