Intermediate ripa lysis buffer

To assess glucose-stimulated insulin secretion (GSIS), β-TC6 cells were incubated in secretion buffer [NaCl 129, KCl 4.8, MgSO₄ 1.2, KH₂PO₄ 1.2, CaCl₂ 2.5, NaHCO₃ 5.0, and HEPES 10 (all mmol/L) supplemented with 1 mg/mL bovine serum albumin, adjusted to pH 7.4] for an additional 60 min with 2.8 or 20 mmol/L glucose [5 (link)]. After collecting the supernatant for insulin measurement, the β-TC6 cells were lysed in intermediate RIPA Lysis Buffer (Beyotime) for later evaluation of the total protein content with BCA protein assay reagent kit (Cat. No. P0010, Beyotime) following the manual. Insulin levels in cell culture medium and β-TC6 cells were measured using a mouse/rat insulin ELISA kit (Linco Research) and standardized by every milligram protein per hour in each well.

Cells were gently washed three times in 1× PBS and then 50 μL of intermediate RIPA Lysis Buffer (Beyotime) was added per 24-well plate. Protein concentrations were detected using bicinchoninic acid (BCA) protein assay kits (Cat. No. P0010; Beyotime). Protein samples (approximately 80 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The primary antibodies used were as follows: Phospho-PERK (3179S, CST, dilution of 1:1000), PERK (3192, CST, dilution of 1:1000), Phospho-eIF2α (9721, CST, dilution of 1:1000), eIF2α (9722, CST, dilution of 1:1000), Caspase-3 (9662, CST, dilution of 1:1000), Parp-1 (9532, CST, dilution of 1:1000), Bcl-2 (4223, CST, dilution of 1:1000), ATF-6 (65880, CST, dilution of 1:1000), IRE1α (3294, CST, dilution of 1:1000), and β-actin (sc-130657, Santa Cruz, dilution of 1:800). The secondary antibody was anti-rabbit (s3738, Promega, dilution of 1:7500) alkaline phosphatase-conjugated antibody. Protein coloration was performed using the Stabilized Substrate for Alkaline Phosphatase (S3841, Promega) and was screened by the FluorChem R system (ProteinSimple, San Jose, CA, USA).