Sc 17852 r

Manufactured by Santa Cruz Biotechnology

Sourced in China, United States

Sc-17852-R is a research-use laboratory instrument. It is designed for use in scientific research applications. The core function of this product is to facilitate specific analysis and data collection processes. No further details on intended use can be provided in an unbiased and factual manner.

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Anti-Pp38 (sc-17852-R,

4 protocols using sc 17852 r

Inhibitors of Inflammatory Signaling Pathways

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The recombinant murine IFN-γ and IL-17 were purchased from PeproTech (Rock Hill, NJ, USA). The STAT1 inhibitor fludarabine (Flu), JAK inhibitor AG-490, NF-κB inhibitor SN50, phosphorylated (p-)p38 inhibitor SB203580, p-ERK1/2 inhibitor PD98059 and also antibody against p-p38 MAPK(Thr180/Tyr182) (sc-17852-R) were all supplied by Santa Cruz Biotechnology, Inc. (Shanghai, China). The antibodies against NOS(pan) (#2977), p-STAT1(Y701) (#7649), p-STAT1(S727) (#8826), p-ERK1/2(Thr202/Tyr204) (#4370) and p65 (#8242) were all purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against STAT1 (#21044-1) was supplied by Signalway Antibody (Baltimore, MD, USA). Antibodies against β-actin (20536-1-AP) and histone H3 (17168-1-AP) were obtained from Proteintech Group (Wuhan, China). The antibody against IκBα (6A920) was purchased from Novus Biologicals (Littleton, CO, USA). Horseradish peroxidase-conjugated goat anti-rabbit (A21020) and goat anti-mouse (A21010) secondary antibodies were both obtained from Abbkine (Redlands, CA, USA).

GAO Q., LIU Y., WU Y., ZHAO Q., WANG L., GAO S., WEN W., ZHANG W., GUO N., ZHOU J, & YUAN Z. (2015). IL-17 intensifies IFN-γ-induced NOS2 upregulation in RAW 264.7 cells by further activating STAT1 and NF-κB. International Journal of Molecular Medicine, 37(2), 347-358.

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Setdb1 Knockdown Immunoblotting Protocol

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The cells were transfected with Setdb1 siRNA for 48 h. Approximately 30 μg protein was separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were probed using the following primary antibodies: NOX4 (1:500; NB110-58849; Novus), beta-Actin (1:2000; CW0096; CWBIO), SETDB1 (1:1000; 11231-1-AP; Proteintech), E2F1 (1:500; sc-193; Santa Cruz Biotechnology), FOXO4 (1:500; sc-5221; Santa Cruz Biotechnology), Lamin B (1:500; sc-6217; Santa Cruz Biotechnology), JNK (1:500; sc-7345; Santa Cruz Biotechnology), p-JNK (1:400; WL01813; WanleiBio), γH2AX (1:000; 2577; Cell signaling technology), p38 (1:500; sc-7972; Santa Cruz Biotechnology), and p-P38 (1:500; sc-17852-R; Santa Cruz Biotechnology). All were used as the manufacturer’s recommendation. The secondary antibodies were horse radish peroxidase-linked anti-mouse, anti-rabbit, or anti-goat IgG for 2 h at room temperature. The membranes were visualized on a Bio-Rad Chemidoc XRS using a Western Bright ECL Kit (Bio-Rad, Berkeley, CA, United States).

Li X., Chen X., Liu Y., Zhang P., Zheng Y, & Zeng W. (2020). The Histone Methyltransferase SETDB1 Modulates Survival of Spermatogonial Stem/Progenitor Cells Through NADPH Oxidase. Frontiers in Genetics, 11, 997.

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Western Blot Analysis of Signaling Pathways

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Western blot analysis was conducted as described previously^{35 (link)}. Briefly, equal amounts of denatured protein from each lysate were separated by 10% SDS polyacrylamide gels. The proteins were electrophoretically transferred to nitrocellulose membranes using a Bio-Rad Trans-Blot Turbo (Hercules, CA, USA). The amounts of phosphorylated p38, ERK1/ERK2, JNK and levels of IκB-α were detected using rabbit polyclonal anti-p-p38 (Thr 180/Tyr 182)-R (sc-17852-R), mouse monoclonal anti-p-ERK1/2 (pT202/pY204.22A) (sc-136521), mouse monoclonal anti-p-JNK (G-7) (sc-6254) and rabbit polyclonal anti-IκBα (C-21)(sc-371) antibody (Santa Cruz Biotech, Dallas, TX, USA), respectively. These antibodies were used at 1:1000 dilution. Immune complexes were visualised by enhanced chemiluminescence. Ponceau S (0.1% in 5% acetic acid) staining was performed to verify total protein loading. Some blots were stripped and re-probed with rabbit polyclonal anti-p38 (C-20) (sc-535, Santa Cruz, used at 1:1000) antibody to confirm equal loading.

Mukaro V.R., Quach A., Gahan M.E., Boog B., Huang Z.H., Gao X., Haddad C., Mahalingam S., Hii C.S, & Ferrante A. (2018). Small tumor necrosis factor receptor biologics inhibit the tumor necrosis factor-p38 signalling axis and inflammation. Nature Communications, 9, 1365.

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Quantification of Liver Protein Phosphorylation

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Liver protein lysates were obtained in the presence of the ice-cold lysis TNG buffer (Tris-HCl 50 mM, pH7.5, NaCl 200 mM, Tween-20 1%, NP-40 0,2%) supplemented with Complete Mini cocktail, PhosSTOP (Roche, Mannheim, Germany), ß-glicerolphosphate 50 mM (SIGMA), 2 mM phenylmethylsulfonyl Fluoride (PMSF, ROCHE, Mannheim, Germany) and 200 mM Na 3 VO 4 (SIGMA). Protein extracts (50-100 mg) were prepared in laemmli's buffer and analysed by 12% polyacrilamide gel electrophoresis and western blot (Gonzalez-Navarro et al. 2013 (link), Martinez-Hervas et al. 2014) (link). The primary antibodies used were: rabbit polyclonal anti-Phospho-p38 (1/200, sc-17852-R, Santa Cruz Biotechnologies), rabbit polyclonal anti-p38 (1/200, sc-535, Santa Cruz Biotechnologies), rabbit polyclonal anti-Phospho-SAPK/JNK (1/200, Thr183/Tyr185, 9251 Cell Signaling), rabbit polyclonal SAPK/JNK (1/200, 56G8, 9258 Cell Signaling) and mouse monoclonal antia-tubulin (1/500, sc-8035, Santa Cruz Biotechnologies). The HRP-conjugated secondary antibodies (1/500, Santa Cruz Biotechnologies) used were: anti-mouse IgG-HRP (sc-2005) and goat anti-rabbit IgG-HRP (sc-2004) . The immunocomplexes were detected with an ECL Plus detection kit (ThermoFisher Scientific, Barcelona, Spain).

Andrés-Blasco I., Herrero-Cervera A., Vinué Á., Martínez-Hervás S., Piqueras L., Sanz M.J., Burks D.J, & González-Navarro H. (2015). Hepatic lipase deficiency produces glucose intolerance, inflammation and hepatic steatosis. The Journal of endocrinology, 227(3).

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