Donkey anti mouse irdye 680cw

The following primary antibodies were employed in study: rabbit anti-GAPDH [1:400; Cell Signaling Technology (CST); Catalog number-5174S)], rabbit anti-LC3B (1:400; CST; 2775S), rabbit anti-beclin1 (1:350; CST; 5174S), rabbit anti-ATG7 (1:400; CST; 8558S), rabbit anti-P62 (1:400; CST; 5114S), anti-cleaved caspase 8 (1:200; CST; 9748), anti-phospho BRAF (1:1000; CST; 2696T), anti-phospho-MEK (1:400; CST; 9154T), Mouse anti-beta actin (1:10,000; Abcam; ab184220), rabbit anti-LAMP-1(1:250; Abcam; ab25630), rat anti-LAMP-2 (1:250; Abcam; ab25631), rabbit anti-Fas L (1:100; Abcam; ab15285), rabbit anti-caspase 3 (1:400; Thermo Fisher; 4331182), rabbit anti-Fas (1:100; Bio legend; 305611), and mouse anti-a2V (Covance, Denver, USA). For isotype-control antibodies, control mouse IgG (R&D Systems) and rabbit IgG isotype (Invitrogen) were used. Secondary antibodies were as follows: goat anti-rabbit IgG-FITC, donkey anti-mouse IgG AF-594, donkey anti-rabbit IgG AF-594 (Invitrogen), rabbit anti-rat IgG-FITC (Abcam), donkey anti-rabbit IRDye-800CW, and donkey anti-mouse IRDye-680 CW (LI-COR Bioscience, Lincoln, NE).

24 h after transfection cells were washed once with 1x phosphate buffered saline (PBS, Thermo Fisher Scientific) and immediately fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich)/1x PBS for 15 min. Cells were washed three times and permeabilized with 0.1% Triton-X-100 (Sigma-Aldrich) in 1x PBS (5 min/wash) and subsequently blocked with Odyssey Blocking Buffer (LI-COR, Lincoln, Nebraska) for 1 h. Incubation with 1:500 diluted primary antibodies (rabbit anti-5-HT₄ #HPA040591; mouse anti-GFP) was carried out in Odyssey Blocking Buffer for 1 h and followed by another three wash cycles with 1x PBS. Then, cells were incubated with secondary antibody solution (1:750 in Odyssey Blocking Buffer; donkey anti-rabbit IRDye 800CW and donkey anti-mouse IRDye 680CW, LI-COR) for 1 h, protected from light. Cells were washed three times with 1x PBS, scanned with a LI-COR Odyssey Infrared Imaging System and analysed by the software provided by the manufacturer. An antibody list is given in the Supplementary Table S6.

Cells were harvested, washed with ice-cold PBS and lysed in Nonident P-40 (NP-40) lysis buffer containing protease and phosphatase inhibitors (Pierce Protein Biology, USA). Protein concentration in the lysate was determined by BCA protein assay (Thermo Scientific, Rockford, IL, USA). The proteins were separated by 4–12% SDS-PAGE and blotted onto PVDF transfer membranes. The membranes were blocked at room temperature for 1 hr in 5% nonfat dry milk in TBS-T followed by incubation with the indicated primary antibody in TBST containing 5% nonfat milk overnight at 4°C. The membranes were washed three times for 10 min with TBST, followed by incubation with Donkey anti-rabbit IRDye-800CW, or Donkey anti-mouse IRDye-680CW secondary antibodies (LI-COR Bioscience, Lincoln, NE) in TBST containing 3% nonfat milk for 1 hr at RT. Fluorescent blots were imaged by Odyssey Infrared Imaging System (LI-COR Biosciences).