Rabbit polyclonal anti gapdh antibody

Rabbit polyclonal anti–TLR4, IκBα, p–IκBα, and anti–IFN–β antibodies were purchased from GeneTex (Irvine, CA) or Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti–GAPDH antibody was purchased from Sigma–Aldrich (St. Louis, MO). Rabbit anti–NF–κB, phosphorylated NF–κB p65 (p–NF–κB), TNF–α, IL–1β, and IL–6 antibodies were purchased from Cell Signaling Technology (Danvers, MA). Rabbit anti–iNOS was purchased from Abcam (Cambridge, MA). Goat anti–rabbit poly–HRP was purchased from Pierce (Rockford, IL). Alexa Fluor 594 anti–rabbit was purchased from Invitrogen (Carlsbad, CA).

Rabbit polyclonal anti‐CBS antibody was purchased from Proteintech (Cat#: 14787‐1‐AP); mouse monoclonal anti‐CBS antibody was purchased from Santa Cruz Biotechnology (Cat#: sc‐133154); mouse monoclonal anti‐acetylated α‐tubulin (lysine‐40) antibody was purchased from Sigma (Cat#: T7451); human polyclonal anti‐CREST antibody was purchased from Antibodies Incorporated (Cat#:15‐234‐0001); mouse monoclonal anti‐MAD1 antibody was purchased from Santa Cruz Biotechnology (Cat#: sc‐137025); mouse monoclonal anti‐Lamin A antibody was purchased from Abcam (Cat#: ab8980); mouse monoclonal anti‐Alpha tubulin antibody was purchased from Proteintech (Cat#: 66031‐1‐Ig); rabbit polyclonal anti‐GAPDH antibody was purchased from Sigma (Cat#: SAB4300645); Alexa Fluor 488‐conjugated goat anti‐mouse IgG (H + L) (Cat#: ZF‐0512), Alexa Fluor 594‐conjugated goat anti‐rabbit IgG (H + L) (Cat#: ZF‐0516), FITC‐conjugated goat anti‐human IgG (H + L) (Cat#: ZF‐0308), HRP‐conjugated goat anti‐rabbit IgG (H+L) (Cat#: ZB‐2301), HRP‐conjugated goat anti‐mouse IgG (H+L) (Cat#: ZB‐2305) were purchased from Zhongshan Golden Bridge Biotechnology.

Cell extracts were prepared with RIPA buffer containing protease and phosphatase inhibitors. Proteins were separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and then treated with the following primary antibodies: anti-b-FGF (1:500) (Upstate Biotechnology, Inc., Lake Placid, NY, USA), anti-HGF (1:1000) (AbCam, Cambridge, UK and anti-αSMA (1:1000) (Sigma Aldrich, Merck KGaA, Darmstadt, Germany). Anti-GAPDH rabbit polyclonal antibody (1:5000) (Sigma Aldrich, Merck KGaA) was used to normalize the protein content. Horseradish peroxide-conjugated goat anti-mouse or goat anti-rabbit secondary antibody complexes were detected by chemiluminescence (Cell Signaling Technology, Beverly, MA, USA). Imaging and densitometric analyses were performed with a UVITEC Mini HD9 acquisition system (Alliance UVItec Ltd., Cambridge, UK).

Model cell lines were seeded at a density of 2.0 x 10⁶ cells per 100-mm culture dish and cultured until they reached 90% confluency. Total protein was extracted using RIPA buffer mixed with protease inhibitor cocktail (Millipore Merck, 539134) and quantified using Qubit^® Protein Assay Kit (Thermo Fisher, Q33211). Proteins were separated by size by running 20 μg of the protein extract in 8% SDS-PAGE and transferred to Immuno-Blot PVDF Membrane (Bio-Rad, 1620174) using the Trans-Blot^® SD Semi-Dry Transfer Cell (Bio-Rad) protocol. Non-specific interactions were blocked by incubating the membrane in TBS-T with 5% non-fat milk overnight at 4°C with constant agitation. To detect human CYB561 protein, an anti-CYB561 rabbit polyclonal antibody (Sigma-Aldrich, HPA014753) was used as the primary antibody and a goat polyclonal anti-rabbit IgG conjugated to horseradish peroxidase (Millipore, 12–348) for the secondary antibody. To visualize protein bands, the ECL^™ Prime Western Blotting Reagent (Cytiva, RPN2236) was used and images captured using the iBright CL100 Imaging System (Invitrogen). A housekeeping protein was also detected using the same blot by mild stripping and re-probing the membrane with an anti-GAPDH rabbit polyclonal antibody (Sigma-Aldrich, G9545-200).