Mbs1499661

Cells were collected and homogenized in radioimmunoprecipitation assay buffer. Total cell protein extracts were quantified and assessed by Western blot as described elsewhere (15 (link)). The following antibodies diluted in blocking solution [phosphate-buffered saline (PBS) and 0.1% Tween 20 containing 5% milk] were used: anti-HSP90 (1:5000; sc13119, Santa Cruz Biotechnology), anti-Flag (1:3000; F7425, Sigma-Aldrich), anti-TMBIM1 (1:1000; MBS1499661, MyBioSource), anti-BAX (1:1000; 06-499, EMD Millipore), anti-MYC (1:3000; ab9106, Abcam), and anti-actin (1:10,000; sc-8432, Santa Cruz Biotechnology). Bound antibodies were detected using peroxidase-coupled secondary antibodies and the enhanced chemiluminescence system.

Flag-RECS1, MYC-RECS1, BAX, ERp57, LAMP-1/2, LGALS1/galectin-1, and LGALS3/galectin-3 proteins were visualized by immunofluorescence. The following antibodies were used: anti-Flag (F7425, Sigma-Aldrich), anti-Flag M2 (F1804, Sigma-Aldrich), anti-TBMIM1 (MBS1499661, MyBioSource), anti-ERp57 (ab13506, Abcam), anti–LAMP-1 (an24170, Abcam), LAMP-2 (ab18528, Abcam), BAX (ab5714, Abcam), anti-LGAL1/galectin-1 (ab25138, Abcam), and LGAL3/galectin-3 (556904, BD Biosciences). All antibodies were diluted 1:1000. We used a sensitive method based on a confined displacement analysis algorithm to calculate colocalization coefficients between Flag-RECS1 and ERp57 (ER), GM130 (Golgi apparatus), and LAMP-1/2 (endosomes/lysosomes) (63 (link)). The colocalization of images was performed as previously described (63 (link)).