Rabbit anti vcam 1

Whole‐cell lysates were prepared in modified RIPA buffer (50 mmol/L of HEPES, 20 mmol/L of pyrophosphate, 25 mmol/L of β‐glycerophosphate, 50 mmol/L of NaF, 5 mmol/L of Na₂MoO₄, 5 mmol/L of EDTA, 150 mmol/L of orthophenanthrol, 1% NP‐40, 2% deoxycholate, and 1% Triton X‐100) in the presence of protease and phosphatase inhibitors. Protein concentrations were quantified using the Pierce BCA Protein Assay (Thermo Scientific, Waltham, MA). Samples (25 μg protein) were denatured, resolved on an 8% SDS‐polyacrylamide gel, and then transferred to an Immuno‐blot PVDF membrane (Amersham Biosciences, Piscataway, NJ). Incubation with primary antibodies (rabbit anti‐VCAM‐1, mouse anti‐ICAM‐1, rabbit anti‐E‐Selectin, and mouse anti‐GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA) was performed for 2 hours, followed by HRP‐linked anti‐rabbit IgG or anti‐mouse IgG (Bio‐Rad, Hercules, CA) secondary antibodies for 40 minutes at room temperature. Immunoreactive bands were visualized by chemiluminescence using an Amersham ECL Prime Western Blotting Detection Kit (GE Healthcare, Pittsburgh, PA). Densitometry was performed using ChemiDoc MP System software (Bio‐Rad).