Anti prohibitin

Cells were lysed in lysis buffer (50 mM Tris–HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 7.5% glycerol; 1% Triton X-100) supplemented with EDTA-free protease inhibitors (Roche). Benzonase was added to minimize nucleic acid contamination. The protein concentration of samples was determined and affinity purification was performed. Eluates were precipitated by sodium deoxycholate, trichloroacetic acid, and acetone. Equivalent protein quantities of lysates and pellets after precipitation were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked using 5% dry milk in tris-buffered saline containing 0.05% Tween-20. Membranes were then probed with selected primary antibodies, followed by the appropriate HRP-conjugated secondary antibodies (Dako, CA, USA). The following monoclonal primary antibodies were used: anti-FLAG-tag (Sigma-Aldrich), anti-BAG2 (Abcam, UK), anti-ATP citrate (Abcam), anti-RANGAP1 (Abcam), antiCLASP-1 (Abcam), anti-Sec5 (Santa Cruz Biotechnology, CA, USA), anti-Sec8 (Biocompare, CA, USA), anti-Prohibitin 2 (Santa Cruz Biotechnology), anti-Angiomotin (Santa Cruz Biotechnology, USA), anti AIF (Abcam), and anti-Prohibitin (Abcam).

Proteins were isolated using RIPA buffer (Pierce) following the manufacturer’s protocol, concentrated using Amicon Ultra 3K centrifugal filter (Millipore), and quantified using the BCA Protein Quantification kit (Pierce). One microgram of protein from K562 whole cell and EVs was loaded on precast 4%–20% Tris-Glycine gel (Life Technologies) and transferred to PVDF membrane. The membrane was blocked using Pierce TBST blocking buffer (cat. no. 37571) for 1 h at room temperature. Primary antibody incubation was performed overnight at 4° at 1:1000 dilutions, while secondary antibodies were used at 1:10000 dilutions. Membranes were developed with Amersham ECL plus Western Blotting Development kit (GE). Anti-fibrillarin (Abcam, cat. no. ab18380), anti-protein disulphide isomerase (Abcam, cat. no. ab2792), and anti-prohibitin (Abcam, cat. no. ab28172) were used as nuclear, endoplasmic reticulum, and mitochondrial marker, respectively. Anti-PDC6I (Abcam, cat. no. ab88743), anti-Tsg101 (Abcam, cat. no. ab83) and anti-transferrin receptor (Abcam, cat. no. ab84036) were used as EV markers. Goat polyclonal to rabbit IgG (Abcam, cat. no. ab6721) and rabbit polyclonal to mouse IgG (ab 6728) were used as secondary antibodies.

Intact cells or membrane fraction (pelleted by centrifuging permeabilized cell suspensions at 10,000 × g for 5 min) were lysed in cold radioimmunoprecipitation assay buffer, (150 mM NaCl, 1.0% (vol/vol) Octylphenoxypolyethoxyethanol, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulphate, and 50 mM Tris (pH 8.0; Sigma)), supplemented with 1 μg/ml protein inhibitors (leupeptin, antipain, and pepstatin) and 1 mM phenylmethylsulfonyl fluoride. Lysed samples and cytosol fractions were used for immunoblotting. Western blot was performed based on instructions of LI-COR (LI-COR Corporation, Nebraska, USA). Primary antibodies used were Anti-BAK (no. 06-536; Millipore) Anti-cyto c (no. 556433; BD Bioscience), Anti-HA (no. 9110; Abcam), Anti-mtHsp70 (no. MA3-028; Thermo Scientific), and Anti-prohibitin (no. ab28172; Abcam), anti-Bax (N-20, sc-493, SantaCruz), anti-MCL-1 (ADI-AAP-240, Enzo life sciences), anti-Bid (no. AF860, R&D systems), anti-Bim (no.2933, Cell Signaling), anti-Bcl-xL (no. 610211, BD transduction), anti-Actin (no.612656, BD transduction), anti-Calnexin (no. ADI-SPA-860, Enzo Life sciences). Detection of bands was performed on a LI-COR Odyssey scanner. ImageJ was used for quantification of the bands.