Gapdh antibody

Utilizing qRT-PCR, the mRNA abundance of PTGS2, COX-2, ST2, IL-6, and IL-33 was assessed. As a loading control, the common GAPDH gene was amplified concurrently. The RIPA lysis buffer with inhibitors of protease and phosphatase was used to recover cellular protein. According to the procedure outlined previously, the protein expressions of ST2, IL-33, p38, COX-2, p-p38, p-p65, p65, p-JNK, JNK, p-ERK, and ERK were evaluated using western blotting. Loading controls were conducted by exploring the blot with an antibody of GAPDH for the concentrated conditioned culture medium. The direct antibodies utilized for WB in this analysis were included ST2 (1; 1,000; PTMab), phosphorylated p38 (1:500; GeneTex), p38 (1:500; PTMab), phosphorylated ERK1/2 (1:500; GeneTex), ERK1/2 (1:500; GeneTex), JNK (1:1,000; GeneTex), phosphorylated JNK (1:1,000; GeneTex), p65 (1:1,000; GeneTex), phosphorylated p65 (1:1,000; GeneTex), PTGS2 (1:1,000; GeneTex) (all from Cell Signaling, Danvers, MA), IL-33 (1:1,000; GeneTex), and GAPDH antibody (1:10,000; Proteintech).

Berberine chloride dihydrate (Berberine hydrochloride, purity: 98%) was obtained from Yuanye Inc. (Shanghai, China) and Ibuprofen (purity: 98%) was obtained from Aladdin (Shanghai, China). Both of them were used directly after purchase. All other solvents were analytically pure. Deionized water was used in this study. All plasmids were supplied by Shanghai GenePharma Co. Ltd (Shanghai, China). Sources of antibodies were as follows: IKKε antibody (Cell SignalingTechnology, 2690), TBK1 antibody (Cell Signaling Technology, 3013), Phospho-TBK1 Ser172 antibody (Cell Signaling Technology, 5483), Phospho-AMPKα Thr172 antibody (Cell Signaling Technology, 2535), Anti-UCP1 antibody (Abcam, ab10983), F4/80 antibody (proteintech, 28463-1-AP), GAPDH antibody (proteintech, 60004-1-Ig), Anti-rabbit IgG (Cell Signaling Technology, 7074 S), Anti-mouse IgG (Cell Signaling Technology, 7076 S), CL488-conjugated Affinipure Goat Anti-Mouse lgG (H + L) (proteintech, SA00013-1), CL488-conjugated Affinipure Goat Anti-Rabbit lgG (H + L) (proteintech, SA00013-2). 3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, ISO and Oil Red O solution (0.5% in isopropanol) were purchased from Sigma-Aldrich (St Louis, MO, USA). Recombinant Murine TNF-α was purchased from Pepro Tech (Rocky Hill, NJ, USA). The BCA protein assay kit was purchased from Beyotime Biotechnology Co., Ltd (Beijing, China).

Cells were observed 2 days after transfection. Thirty minutes before imaging, the culture medium was changed to FluoroBrite medium (ThermoFisher) with 40 μM DFHBI-1T and 0.1 μg/ml Hoechst. Live cell fluorescence images were acquired on a Nikon microscope.
Western blotting
Cells were lysed in 2X SDS loading buffer (200 mM β-mercaptoethanol, 100 mM Tris pH 6.8, 20% glycerol, 4% SDS, 0.05% bromophenol blue). The lysates were separated by SDS-PAGE and transferred onto the NC membrane, followed by blocking with 5% milk in TBST solution and incubation with primary antibody overnight at 4 °C and secondary antibodies for 1h at room temperature. Finally, the NC membrane was incubated with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and imaged by Gel Imager System. Antibodies included Anti-HA-tag antibody (MBL, M180-3) for dLbCas12a-p300, dLbCas12a-VPR, dLbCas12a-SunTag, scFv-sfGFP-VP64, scFv-sfGFP-VPR, LbCas12a, dAsCas12a-VPR, AsCas12a, and CasRx. Anti-Tubulin antibody (Proteintech, 66240), GAPDH antibody (Proteintech, 60004), and HRP-conjugated horse anti-mouse IgG secondary antibody (CST 7076S).

The western blot assay was performed as described previously (Yu et al., 2016 (link)). NSCs and MSCs were prepared using RIPA buffer. The lysate protein concentrations were determined using a BCA protein assay kit (Beyotime Biotechnology, Nanjing, China). The protein samples were denatured in Laemmli buffer at 100°C for 10 min before electrophoresis, and were then subjected to 10% SDS-PAGE before being electrotransferred to polyvinylidene difluoride membranes with a standard transfer solution. After blocking with 10% nonfat milk, membranes were incubated with primary antibodies against β-tubulin III (1:1,000; Abcam, Cambridge, MA), MAP2 (1:1,000; Abcam, Cambridge, MA), Runx2 (1:1,000; Abcam, Cambridge, MA), BMP-2 (1:2000; ProteinTech Group), and OPN (1:2000; ProteinTech Group), and GAPDH antibody as a control (1:5,000; ProteinTech Group). Proteins were visualized by chemiluminescence using an ECL kit (Share-Bio, Jinan, China).

Total protein lysates and plasma membrane proteins were extracted from HEK293 cells co-transfected KChIP2 with wild type or mutant K_v4.3 after 48 hours following a standard protocol as previously described [38 ]. Plasma membrane proteins were isolated by the Pierce Cell Surface protein Isolation Kit (Thermo Scientific, Waltham, MA, USA). The total proteins or plasma membrane proteins were subjected to western blot analysis using a polyclonal anti-K_v4.3 antibody (Alomone, Jerusalem, Israel) or GAPDH antibody (Proteintech Company, Wuhan, China).