Dc reagent

Cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1% v/v Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM sodium glycerol phosphate, 1 mM sodium orthovanadate, 1 mM PMSF, and 5 μg/ml each of antipain, pepstatin and leupeptin. The lysates were sonicated and clarified by centrifugation at 16,000 g for 10 min at 4°C. Total protein in cell lysates was estimated by Bio-Rad DC Reagent following manufacturer’s instructions. One hundred μg of total protein was resolved by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted using a primary antibody, followed by HRP-conjugated secondary antibody diluted in Tris-buffered saline containing 5% w/v non-fat dried milk and 0.1% v/v Tween-20. Enhanced chemiluminescent lighting system, PerkinElmer Life Sciences (Boston, MA) was used for detection.
For immunoprecipitation, Nalm6 cells were treated with DMSO or ANX2T inhibitor for 6 h prior to lysis as described above. Lysates corresponding to 1 mg of total protein were incubated overnight with anti-p11 antibody (25 μg per sample) coupled to Protein A Mag Agarose beads, GE^® Biosciences (Pittsburg, PA), via rabbit anti-mouse antibody. The beads were washed and the proteins bound to the beads were resolved on SDS-PAGE and detected by immunoblotting as described above.

Liver samples were homogenised in buffer consisting of 10 mM Tris–HCl pH 7.5, 1 mM EGTA, 75 mM sucrose, 225 mM sorbitol and 0.1% BSA, before lysis at 4°C in 50 mM Tris–HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS and 1 × protease inhibitor cocktail (Roche). Protein concentration was determined by Lowry assay (DC Reagent, Bio-Rad), and 5 μg of protein resolved on PAGE gels (Novex) before transfer to Imobilon-P membrane (Millipore). Following blocking with 5% non-fat dry milk in PBS with 0.1% (v/v) Tween-20, membranes were incubated overnight with the following primary antibodies: mouse anti-NDUFB8 (1:10 000, Abcam AB110242), mouse anti-COX IV (1:10 000, Abcam AB14744), rabbit anti-MPV17 (1:1000, Proteintech 10310-1-AP) or rabbit anti-TOM20 (1:20 000, Santa Cruz Biotechnology SC-11415). Membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:4000, Promega) in 5% non-fat dry milk in PBS with 0.1% (v/v) Tween-20, and visualised using enhanced chemiluminescence (ECL, G.E. Healthcare).

Cells were lysed with RIPA buffer and ultrasound. Protein concentration was measured with DC reagent (Bio-Rad Laboratories, Munich, Germany). Equal amounts of protein were loaded on a 4%–20% tris-glycine gel (Novex, Life Technologies, Carlsbad, CA, USA) and separated by SDS-PAGE. Proteins were transferred by semi dry blot on a PVDF membrane (Immobilon-FL; Millipore/Merck, Darmstadt, Germany). For 2nd antibody, goat-anti-rabbit-IRDye800CW and goat-anti-mouse-IRDye680 (LI-COR, Bad Homburg, Germany) were used. Blots were dried and scanned using an Odyssey infrared imager (LI-COR, Bad Homburg, Germany). Used antibodies: β-Actin (CS, A5441), α-Tubulin (Epitomics, 1878-1), PARP (CS, 9542S), p21 (CS, 2946), p27 (CS, 2552), p15 (CS, 4822).

Protein fractionation, transfer and immuno-detection were performed as described (Dalla Rosa et al., 2014 (link)), with some modifications. Muscle and liver samples were prepared as previously described (Cooper et al., 2003 (link)). Cells were lysed on ice in phosphate-buffered saline (PBS), 0.1% n-dodecyl β-d-maltoside (DDM), 1% SDS, 1 × protease inhibitor cocktail (Roche), 50 U Benzonase® and phosphatase inhibitor complexes (Cell signalling), or in RIPA buffer containing 1× protease inhibitor cocktail (Roche). Protein concentration was measured by Lowry assay (DC^™ Reagent, Bio-Rad) or BCA assay, and 10 μg of lysate analysed per lane. Primary antibodies were: mouse anti-GAPDH (1:20 000, Abcam), mouse anti-NDUFB8 (1:1000, Abcam), mouse anti-COX II (1:2000, Abcam), mouse anti-VDAC1 (Porin) (1:10000, Merck), rabbit anti-ATAD3 (1:60 000, gift from John Walker), rabbit anti-SREBF2 (1:1000, Abcam), rabbit anti-CES-1 (1:2000, Proteintech).

T- lymphoblasts and EBV-transformed B-cells were washed in PBS, pelleted by centrifugation, and lysed in Frackeltons buffer containing protease and phosphatase inhibitors. Lysate protein concentration was determined using a DC reagent (Bio-Rad). Samples (25 μg) in Laemmli Buffer were separated on acrylamide gels (10%) at 75 V for 45 min followed by 120 V and transferred to PVDF membranes at 110 V for 90 min, or 40 V overnight at 4℃. Membranes were washed in TBST, blocked in 5% milk, and incubated in primary antibody overnight at 4℃. The following primary (STIM1—1:1000, Cell Signaling Technology, #5668; β-actin—1:5000, Sigma Aldrich, #A1978; HSP90α/β—1:5000, Santa Cruz Biotechnology, #sc-13119) and secondary (anti-Mouse HRP—BD Biosciences, #554002; anti-Rabbit HRP—Bio-Rad, #172–1019) antibodies were used.