Anti gapdh igg

Thioglycolate-elicited peritoneal macrophages were seeded in 10% FBS containing DMEM and incubated overnight. Unadhered cells were washed off, and 1% bovine serum albumin (BSA) containing DMEM was added to the plate and incubated for 3 h before ginkgetin treatment. To determine expression of CD36, TRPV4, TLR2, TLR4, TLR6, and GAPDH proteins whole cell lysates were prepared from cells treated overnight with ginkgetin (1 and 5 µM) or vehicle in the presence or absence of oxLDL (25 µg/ml). To determine the expression levels of LPS-triggered p-JNK and JNK, cells were pretreated with ginkgetin (5 and 10 µM) for 3 h and then stimulated with E. coli LPS for 30 min. Whole cell lysates were prepared from twice-washed cells (ice cold PBS) using RIPA buffer with added protease-inhibitor cocktail (Thermo Scientific, MA). Blots were probed with rabbit anti-TRPV4 (Alomone Lab, Jerusalem, Israel), anti-mouse CD36 (R&D, Minneapolis, MN), rabbit anti-TLR4, rabbit anti-TLR6, rabbit anti-JNK, and rabbit anti-p-JNK primary antibodies (Cell Signaling, Danvers, MA) followed by anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (R&D, Minneapolis, MN). Blots were stripped and re-probed with anti-GAPDH IgG (1:2000; Santa Cruz, Dallas, TX) for loading control.

Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (Dublin, IRL). Cytokines (TNF-α, IL-6), apocynin and NSC23766 were purchased from Millipore (Cork, IRL). Primary antisera were purchased from the following sources: Anti-occludin IgG, anti-claudin-5 IgG, and anti-ZO-1 IgG (Bio-Sciences, Dublin, IRL); Anti-VE-cadherin IgG (Abcam, Cambridge, UK); Anti-gp91 IgG, anti-p47 IgG, and anti-GAPDH IgG (Santa Cruz Biotechnology, CA, USA); HRP-conjugated secondary antisera for VE-cadherin, occludin, claudin-5, and GAPDH were purchased from Cell Signalling Technologies Inc. (MA, USA). HRP-conjugated secondary antisera for gp91 and p47 were purchased from Sigma Aldrich. siRNA constructs for gp91 (SC35503, RefSeq NM_000397.3) and p47 (SC29422, RefSeq NM_000265.5) were obtained from Santa Cruz Biotechnology.