Alexa fluor 488 conjugated goat anti mouse

The ER stress activators thapsigargin (TG) and tunicamycin (TM) were purchased from Sigma-Aldrich; Merck KGaA and Beijing Solarbio Science & Technology Co., Ltd., respectively. The ER stress inhibitors 4-phenylbutyric acid (PBA) and sodium tauroursodeoxycholate (TUDCA) were purchased from Sigma-Aldrich; Merck KGaA. TM, TG, PBA and TUDCA were dissolved in dimethyl sulfoxide (DMSO; Leagene). Anti-glucose-regulated protein 78 kDa (GRP78; ab12223), anti-activating transcription factor (ATF)6 (ab11909), anti-phosphorylated eukaryotic initiation factor 2α (p-IRE1α; ab48187), anti-E-Cadherin (ab40772), anti-fibronectin (ab2413) and anti-α-SMA (ab32575) primary antibodies were purchased from Abcam. Horseradish peroxidase-conjugated anti-p-eIF2α (119A11), horse anti-mouse and horse anti-rabbit secondary antibodies, Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 488-conjugated goat anti-mouse secondary antibodies were purchased from Cell Signaling Technology, Inc. Anti-ATF4 primary antibody (sc-390063) was purchased from Santa Cruz Biotechnology, Inc. Primary antibodies against vimentin (10366-1-AP), β-actin (66009-1-Ig) and Neural cadherin (N-cadherin; 22018-1-AP) were purchased from ProteinTech Group, Inc.

VSMCs (1x10⁵ cells/ml) were seeded in 24-well climbing slice culture plates treated with 10 mmol/l β-GP for 3 days at 37˚C following plasmid transfection. They were then fixed in 4% paraformaldehyde for 1 h at RT. Following washing with PBS, cells were permeabilized using 0.5% Triton X-100 for 10 min at RT. Next, cells were washed with PBS and blocked with 5% bovine serum albumin (cat. no. A8850-5; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at RT. The cells were then double-stained with primary antibodies against BMP2 (1:100; cat. no. ab214821; Abcam) and α-SMA (1:100; cat. no. 48938; Cell Signaling Technology, Inc.) in PBS overnight at 4˚C. On day 2, following extensive washing with PBS, cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse (1:200; cat. no. ZF-0511; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) or Alexa Fluor 594-conjugated goat anti-rabbit (1:200; cat. no. ZF-0516; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) for 1 h at RT. Following staining with DAPI for 10 min at 37˚C, cells were viewed by fluorescence microscopy (magnification, x200; Nikon Eclipse NI-E; Nikon Corporation) and analyzed using ImageJ Software (version 1.48; National Institutes of Health).