Ab74139

For determination of SDC2/4 protein expression levels in vitro, control and LPS group cells were seeded in 6-well plates. Cell lysates were prepared using RIPA lysis buffer with protease inhibitors. After determination of the overall protein concentrations using a bicinchoninic acid protein assay, 50 μg lysate samples were loaded per lane and separated by 12.5% sodium dodecyl gel electrophoresis for approximately 1 h. Proteins were subsequently transferred to membranes for 2 h and blocked in milk plus 5% bovine serum albumin (1 h). Primary antibodies were rabbit anti-SDC2 (1:1000, Abcam, ab205884) and rabbit anti-SDC4 (1:1000, Abcam, ab74139). Mouse anti-tubulin was used as an endogenous control. Membranes were incubated with primary antibodies overnight in a 4 °C refrigerator and then washed three times for 10 min, followed by the addition of horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:10000, Abcam, ab288151) and incubation at room temperature (1 h). Finally, specific protein bands were visualized using an enhanced bioluminescence instrument (Bio-Rad, USA) under ECL reagent. Each experiment was conducted in triplicate.