Goat anti rabbit igg h l hrp

The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and Akt agonist SC79 were purchased from Selleck (Houston, United States). Rabbit monoclonal anti-LGALS14 (ab150427), rabbit monoclonal anti-VE-cadherin (ab33168), rabbit monoclonal anti-N-cadherin (ab76011), mouse monoclonal anti-MMP-9 (ab119906), rabbit monoclonal anti-TIMP1 (ab109125), rabbit monoclonal anti-MMP-2 (ab92536), rabbit monoclonal anti-TIMP-2 (ab180630), rabbit monoclonal anti-GAPDH (ab181602), rabbit monoclonal anti-cytokeratin 7 (ab181598), and mouse monoclonal anti-HLA G (ab52455) antibodies were purchased from Abcam (Cambridge, United Kingdom). Rabbit monoclonal anti-p-Akt (Ser473) (9271) and rabbit monoclonal anti-Akt (9272) antibodies were purchased from Cell Signaling Technology (Danvers, United States). Goat anti-mouse IgG (H + L) HRP and goat anti-rabbit IgG (H + L) HRP secondary antibodies were purchased from MultiSciences Biotech Co. (Hangzhou, China).

The western blot procedure is described in detail in our previous study^{11 (link)}. The blots were cut prior to hybridization with primary antibodies during blotting. Primary antibodies included anti-OPG (#sc-390518, Santa Cruz Biotechnology), anti-DUSP14 (#ab272587, Abcam), anti-extracellular signal-regulated kinase (ERK, # 4695, Cell Signaling Technology), anti-phospho-ERK (#4370, Cell Signaling Technology), anti-P38 (#9212, Cell Signaling Technology), anti-phospho-P38 (#4511, Cell Signaling Technology), anti-c-Jun NH2-terminal kinase (JNK, #9252, Cell Signaling Technology), anti-phospho-JNK (#4668, Cell Signaling Technology), and anti-β-actin (#TA-09, ZSGB-BIO, China) antibodies. Secondary antibodies included Goat Anti-Rabbit IgG(H + L) HRP (GAR007, MULTI SCIENCES, China) and Goat Anti-Mouse IgG(H + L) HRP (GAM007, MULTI SCIENCES, China).

Cell culture medium was collected, cells were lysed, total protein was extracted, and protein was separated using 8% and 12% SDS-PAGE. The proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane, and blocked with 5% skim milk for 1.5 h at room temperatureat. Blots were incubated with the primary antibodies overnight, washed, incubated with secondary antibodies for 1.5 h, and washed again. Enhanced chemiluminescence (ECL) was used for color development and exposure. Bands detected using WB were scanned in grayscale mode using a multifunction imager (Thermo Fisher Scientific, Waltham, MA, USA). The protein expression of HIF-1α, Beclin-1, and LC3 was then detected. Finally, ImageJ software was used to quantify the WB bands. The antibodies used in this experiment were as follows: HIF-1α (1:1000; Affinity), Beclin-1 (1:1000; Affinity), LC3A/B (1:1000; CST), and GAPDH (1:3000; ZENBIO), Goat Anti-Rabbit IgG(H+L) HRP (1:20000; MULTISCIENCES)