Journal: Scientific Reports

Article Title: The inhibition of Bax activation-induced apoptosis by RasGRP2 via R-Ras-PI3K-Akt signaling pathway in the endothelial cells

doi: 10.1038/s41598-019-53419-4

Figure Lengend Snippet: Activation of R-Ras-PI3K-Akt signaling pathway by RasGRP2 and suppression of apoptosis via its signaling pathway. ( a – e ) Each effect was detected by western blotting. ( a ) Activated R-Ras and Rap2A were isolated by pull-down assay using RalGDS-RBD agarose beads. ( b ) Activated TC21 was isolated by pull-down assay using Raf1-RBD agarose beads. ( c – e ) Phosphorylation of Akt was detected using specific antibodies. Cells were treated with siRNAs against R-Ras or negative control siRNA ( d ), or incubated with or without LY294002 ( e ). ( f , g ) Cell viability was determined by Cell Counting Kit-8 assay. ( f ) Cells were pre-treatment with siRNAs against R-Ras or negative control siRNA and incubated with or without BAM7 or anisomycin for 48 h. ( g ) Cells were pre-incubated with or without LY294002 and incubated with or without BAM7 or anisomycin for 48 h. M: mock cells, R: RasGRP2-stable overexpression cells, aniso: anisomycin. Data are shown as the mean ± SD (n = 3), ** P < 0.01 compared with each cell control, ## P < 0.01 compared with each R cell without pre-treatment or transfected with the negative control siRNA, and ++ P < 0.01 compared with each M cell transfected with the negative control siRNA.

Article Snippet: After washing with PBS containing 0.05% Tween 20 (PBS-T), the membranes were incubated with rabbit anti-PARP antibody (GeneTex, GTX100573 at 1:1,500), rabbit anti-RasGRP2 antibody (GeneTex, GTX108616 at 1:5,000), mouse anti-β-actin antibody (Santa Cruz, sc-47778 at 1:12,000), rabbit anti-Rap1A antibody (Santa Cruz, sc-398755 at 1:1,000), mouse anti-p-Akt (Thr308) antibody (Santa Cruz, sc-271966 at 1:6,000), mouse anti-p-Akt (Ser473) antibody (Santa Cruz, sc-81433 at 1:500), mouse anti-Akt antibody (Santa Cruz, sc-81434 at 1:2,000), rabbit p-JNK antibody (Cell Signaling Technology, #4671 at 1:2,000), rabbit JNK antibody (Cell Signaling Technology, #9258 at 1:2,500), mouse anti-R-Ras antibody (Santa Cruz, sc-166221 at 1:100), mouse anti-TC21 antibody (Santa Cruz, sc-166262 at 1:100), rabbit anti-Rap2A antibody (GeneTex, GTX108831 at 1:500), mouse anti-Bax antibody (Santa Cruz, 2D2: sc-20067 at 1:200), rabbit anti-α-tubulin 4a antibody (GeneTex, GTX112141 at 1:1,000), mouse anti-VDAC-1 antibody (Santa Cruz, sc-390996 at 1:1,000), mouse anti-cytochrome c antibody (Santa Cruz, sc-13156 at 1:500), rabbit anti-MCL1 antibody (GeneTex, GTX102026 at 1:500), rabbit anti-Hexokinase 2 antibody (GeneTex, GTX111525 at 1:5,000) or rabbit anti-Hexokinase 1 antibody (GeneTex, GTX105248 at 1:3,000) in Can Get Signal® Solution 1 (Toyobo) for 1 h. Subsequently, the membranes were washed thrice with PBS-T and incubated with anti-rabbit IgG antibody (GeneTex, GTX77057 at 1:5,000) or anti-mouse IgG antibody (DakoCytomation, P0260 at 1:5,000) in Can Get Signal® Solution 2 (Toyobo) for 1 h. After five additional washes with PBS-T, immunoreactive proteins were detected using Western BloT Quant HRP or Ultra Sensitive HRP Substrate (Takara) and Amersham hyperfilm ECL (GE Healthcare).

Techniques: Activation Assay, Western Blot, Isolation, Pull Down Assay, Phospho-proteomics, Negative Control, Incubation, Cell Counting, Over Expression, Control, Transfection