Anti β actin antibody ac 15

Western blotting analysis was performed as previously described^{20 (link)}. Following antibodies were used: anti-PU.1 antibody (D-19), anti-IRF4 antibody (M-17), and anti-IRF8 antibody (C-19), (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-β-actin antibody (AC-15, Sigma-Aldrich).

Dissected aortas were homogenized with buffer containing 50 mmol/L Tris‐HCl (pH7.4), 150 mmol/L NaCl, 1% Nonidet P‐40, 1 mmol/L EDTA, 1% protease inhibitor cocktail (P8340) (Sigma‐Aldrich, Darmstadt, Germany), and 1% phosphatase inhibitor cocktail (167‐24381) (Wako Pure Chemical Industries, Osaka, Japan). The homogenate containing 100 μg (for α₁ adrenergic receptor) or 50 μg protein (for eNOS) was separated by 6% SDS‐polyacrylamide gel electrophoresis, and transblotted to PDVF membrane (Bio‐Rad, Hercules). α₁ Adrenergic receptor was detected by ab3462 (Abcam, Cambridge, UK) and eNOS was detected by 610296 (BD Science, Franklin Lakes) as a primary antibody, respectively. The membranes were striped and reincubated with anti‐β‐actin antibody (AC‐15) (Sigma‐Aldrich, St Louis) for normalization. Signals were detected by horse radish peroxidase‐conjugated secondary antibodies and ECL select (GE Healthcare Life Science, Buckinghamshire, UK). The density of the signals was then quantified by LAS 4000mini using ImageQuant TL software (GE Healthcare Life Science Buckinghamshire, UK).

Bleomycin was generously donated by Dong-A ST Co., Ltd. (Seoul, Korea), while mirodenafil was generously donated by SK Chemicals Co., Ltd. (Seongnam, Korea). Anti-α-smooth muscle actin (SMA) antibody (MAB1420) was purchased from R&D Systems. Anti-COL1A1 antibody (3G3) was purchased from Santa Cruz Biotechnology. Anti-phospho-Smad2/3 antibody (D27F4), anti-Smad2/3 antibody (D7G7), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (2118S) were purchased from Cell Signaling Technology. Anti-β-actin antibody (AC-15) was purchased from Sigma-Aldrich. Recombinant human and mouse TGF-β1 proteins were purchased from R&D Systems.

Western blot was performed as described previously [47 (link)],[48 (link)]. NFκB p65 plasmid DNAs (2 μg/35 mm dish) were transfected into HEK293 cells and SH-SY5Y cells. 48 hours after transfection, cells were harvested by RIPA-DOC buffer (1% Triton X-100, 0.1% sodium dodecylsulphate, 1% sodium deoxycholate, 0.15 mM NaCl, 0.05 M Tris–HCl pH 7.2, 0.5 mM phenylmethylsulfonyl fluoride) containing protease inhibitors (Roche). Cell lysates were separated on 10% Tris-glycine gel and proteins were transferred onto PVDF membrane, followed by primary antibody incubation overnight at 4°C. The primary antibodies were rabbit anti-PINK1 polyclonal antibody (1:500 dilution, Novus BC100-494), anti-p65 (1:1000 dilution, Cell Signaling), monoclonal anti-NFκB p65 antibody (1:1000 dilution, Sigma), and anti-β-actin antibody AC-15 (1:100, 000 dilution, Sigma). The protein bands were visualized using the LI-COR Odyssey (LI-COR Biosciences) after secondary antibody (goat anti-mouse, goat anti-rabbit, LI-COR Biosciences) labeling. The resulted protein bands were quantified by Image J software. The experiment was done in triplicate to minimize experimental errors.

Western blotting was performed as previously described (39 (link)). Anti-NLRP3 antibody (D4D8T, Cell Signaling Technology, Danvers, MA, USA), anti-Caspase-1 antibody (EPR19672, Abcam, Cambridge, UK), anti-ASC antibody (B-3, Santa Cruz Biotechnology), anti-PU.1 antibody (T21, Santa Cruz Biotechnology), anti-IRF8 antibody (C19), and anti-β-actin antibody (AC-15, Sigma-Aldrich, St. Louis, MO, USA) were used as primary antibodies.