Rabbit igg isotype

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).

Subcellular fractionation, immunoprecipitation analysis, and immunoblotting were performed as described previously (Semenas et al., 2014 (link)). Briefly, protein from different subcellular fractions was prepared by using Subcellular Protein Fractionation Kit for Cultured Cells (Cat#78840, Thermo Scientific™) according to the manufacturer’s protocol. For immunoprecipitation analysis, the Protein G Sepharose™ 4 Fast Flow beads (Cat#17-0618-01, GE Healthcare) and anti-PIP5K1α antibody (Cat#15713-1-AP, Proteintech) were applied to pull down PIP5K1a from the protein lysates. The rabbit IgG isotype (Cat#02-6102, Invitrogen) was used as a control. For immunoblotting, antibodies against β-tubulin (Cat#075K4875, Sigma-Aldrich) and Lamin B (Cat#sc-6216, Santa Cruz) were used, respectively, as controls for cytoplasmic and nuclear fractions. The signal was captured and documented with the Proxima C16 Phi+ imaging system (Isogen Lifescience). Densitometric quantification of immunoblots was performed by the software ImageJ 1.50i Software (NIH, Baltimore, MD, Unites States) and represented as fold change relative to control and was normalized relative to actin or GAPDH bands.