Pierce bca protein assay kit

The transfected HCC cells were lysed by protein lysis buffer (Beyotime, Shanghai, China) and the concentrations of protein was measured using a Pierce BCA protein assay kit (Tiangen Biotech Co., Ltd., Beijing, China). A total of 20‐30 μg protein was separated by 10% SDS‐PAGE and electro‐transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After being blocked with 5% skimmed milk, the membranes were incubated with VGLL4 (ab140290; Abcam, Cambridge, MA, USA) and GAPDH antibodies (ab181602; Abcam) overnight at 4°C. After incubation with the horseradish peroxidase‐conjugated IgG secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The protein expression levels were detected by ECL reagent (Millipore) and imaged using Amersham Imager 600 from GE Healthcare Life Sciences (Beijing, China). The blots were semi‐quantified by ImageJ software (1.46; National Institutes of Health, Bethesda, MD, USA).

To measure the protein level of galectin-3 in both corneal epithelial cells and tissues from patients with fungal keratitis, cellular and tissue lysates were prepared by lysing samples in ice-cold RIPA buffer (25 mM Tris-HCl pH 7.6, 0.15 M NaCl, 1% IGEPAL CA-630, 1% sodium deoxycholate, 0.1% SDS) containing 1%PMSF. Protein extraction was precisely performed using a Pierce BCA Protein Assay Kit (Tiangen, Beijing, China). For each sample, 40 μg of protein was loaded into each well. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 12% gels to separate proteins. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen, USA), which was then blocked for 2 h with 5% bovine serum albumin (BSA). The membrane was then incubated with rat anti-mouse galectin-3 mAbs (Abcam, UK) overnight at 4°C. On the next day, the membrane was washed and incubated with anti-rat or peroxidase-conjugated secondary antibodies (Abcam, UK) for 1 h. Immuno-reactive bands were visualized in an Odyssey Infrared Imaging System (LI-COR Biosciences, Frederick, MD) according to the supplier's instructions. GAPDH was used as the loading control. The integrated density of bands in the obtained results was further quantitatively analyzed by ImageJ.

The transfected CRC cells were lysed by protein lysis buffer (Beyotime, Shanghai, China) and the concentrations of protein was measured using a Pierce BCA protein assay kit (Tiangen Biotech Co., Ltd., Beijing, China). A total of 20–30 μg protein was separated by 10% SDS‐PAGE and electro‐transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). After being blocked with 5% skimmed milk, the membranes were incubated with HMGB3 (Wuhan Sanying 27465-1-AP) and GAPDH antibodies (ab181602; Abcam) overnight at 4°C. After incubation with the horseradish peroxidase‐conjugated IgG secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, United States). The protein expression levels were detected by ECL reagent (Millipore) and imaged using Amersham Imager 600 from GE Healthcare Life Sciences (Beijing, China). The blots were semi‐quantified by ImageJ software (1.46; National Institutes of Health, Bethesda, MD, United States) Tissue samples were fixed in 4% paraformaldehyde embedded in paraffin, and sectioned. The tissue sections were incubated with HMGB3 primary anti-bodies at 4°C overnight and then incubated with an HRP-conjugated secondary antibody.