Goat anti rabbit igg h l hrp conjugated antibody

cells were infected with viruses at MOI 10 and cell lysates were harvested at various time points (HOS cells, n = 3, A549 cells, n = 2, biological replicates). Western blot was performed as described above. The primary antibodies used were: rabbit polyclonal anti-LC3B (clone: PA1-16930, Thermo Fisher Scientific) and rabbit polyclonal anti-SQSTM1/p62 (clone: 5114, Cell Signaling Technology, Danvers, MA). The goat anti-rabbit IgG(H + L)-HRP conjugated antibody (clone: G-21234, Thermo Fisher Scientific) was used as the secondary probe. The intensity of the LC3-I and LC3-II bands was quantified with Image J

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.

For protein detection, cells were harvested and lysed 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, 1 mM EDTA] supplemented with protease and phosphatase inhibitors plus 1 mM DTT (Sigma Aldrich, Stockholm, Sweden) as described previously [18 (link)]. Thirty µg total protein were loaded into each well of a NuPAGE 4–12% Bis-Tris gradient gel (ThermoFisher, Stockholm, Sweden) and subjected to electrophoresis. The proteins were then transferred to a Hybond low-fluorescent 0.2 µm PVDF membrane (Amersham, Buckinghamshire, UK), blocked for 1 h in Odyssey^® Blocking Buffer (PBS) (LI-COR), and stained overnight at 4°C with antibodies against NLRP12 (Abcam, Stockholm, Sweden) and GAPDH (ThermoFisher, Stockholm, Sweden) as loading control. The membranes were then probed with the goat anti-rabbit IgG (H+L) HRP-conjugated antibody (ThermoFisher, Stockholm, Sweden) or the goat anti-mouse IRDye 680RD antibody (LI-COR Biotechnology, Lincoln, NE, USA) and proteins were detected using Clarity™ ECL substrates (BioRad, Hercules, CA, USA) and Super RX-N film (FujiFilm Nordic AB, Stockholm, Sweden), or the LI-COR Odyssey^® CLx scanner operating with Odyssey^® Image Studio software (LI-COR Biotechnology).