Sab4200130

For protein detection, 1.0 ×
10⁶ cells were seeded and exposed to silica NPs at 2.5
μg/mL with or without pre-incubation with the specified inhibitors.
The cells were then collected and lysed overnight at 4 °C in
RIPA buffer [50 mM Tris HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100,
0.25% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA]. Protease and
phosphatase inhibitors (Mini EDTA-free Protease Inhibitor Cocktail,
Sigma-Aldrich; 1 mM PMSF, Thermo Fisher; PhosSTOP, Sigma-Aldrich)
and 1 mM DTT (Sigma-Aldrich) were freshly added to the buffer. Cell
lysates were centrifuged at 13.000g for 20 min, and
supernatants were collected. The protein concentration was measured
by Bradford assay, and 30–50 μg of protein was loaded
into each well of a NuPAGE 4–12% Bis-Tris gradient gel (Thermo
Fisher) and subjected to electrophoretic separation. The proteins
were then transferred to a Hybond Low-Fluorescent 0.2 μm PVDF
membrane (Amersham), blocked for 1 h in Odyssey Blocking Buffer (PBS;
LI-COR), and stained overnight at 4 °C with primary antibodies
against iPLA₂ (Sigma-Aldrich, SAB4200130). Membranes were
reprobed for GAPDH (Thermo Fisher). The goat antirabbit IgG (H+L)
HRP-conjugated antibody (Thermo Fisher Scientific) or goat antimouse
IRDye 680RD antibody (LI-COR) was used as a secondary antibody. The
proteins were analyzed on the LI-COR Odyssey CLx scanner using Odyssey
Image Studio software.