Protein expression was analyzed by western blotting as previously described (Chai et al. 2014 (link); Yuan et al. 2015 (link)). For western blot analysis, primary antibodies against NFκB (rabbit polyclonal antibody, ab16502), TNFα (rabbit polyclonal antibody, ab6671), NLRP3 (rabbit polyclonal antibody, ab210491), IL-1β (rabbit polyclonal antibody, ab9722), IL-6 (rabbit polyclonal antibody, ab208113) and caspase1 (rabbit polyclonal antibody, ab1872) were purchased from Abcam (UK). TLR4 (mouse monoclonal antibody, sc-293072) and ASC (mouse monoclonal antibody, sc-271054) were purchased from Santa Cruz (USA). IL-18 (rabbit polyclonal antibody, TA324190) was purchased from ORIGENE (USA). The goat anti-mouse IgG-HRP (ZB2305) and goat anti-rabbit (ZB2301) IgG-HRP secondary antibodies were purchased from ZSGB-Bio, Beijing, China. The targeted proteins were visualized with the Super Signal West Femto Chemiluminescent Substrate (Thermo Scientific Pierce) and the intensity of the visualized bands were analyzed using Quantity One software (Bio-Rad). β-actin (mouse monoclonal antibody, TA-09, Zhongshan Jinqiao Biotech company, Beijing, China) was used as an internal control. Data are expressed as a ratio to β-actin.
Goat anti mouse igg hrp zb2305
Manufactured by ZSGB-BIO
Sourced in China
Goat anti-mouse IgG-HRP (ZB2305) is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to bind to mouse immunoglobulin G (IgG) antibodies and can be used in various immunoassay and immunodetection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Goat anti rabbit zb2301 igg hrp secondary antibodies, by ZSGB-BIO (2 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg hrp zb 2301, by ZSGB-BIO (2 mentions)
Ab1872, by Abcam (1 mentions)
Ab210491, by Abcam (1 mentions)
Ab6671, by Abcam (1 mentions)
Ab208113, by Abcam (1 mentions)
Sc 271054, by Santa Cruz Biotechnology (1 mentions)
Ta324190, by OriGene (1 mentions)
Ab16502, by Abcam (1 mentions)
Ab9722, by Abcam (1 mentions)
Sc 293072, by Santa Cruz Biotechnology (1 mentions)
Mettl14 hpa038002, by Merck Group (1 mentions)
Tenascin c ab108930, by Abcam (1 mentions)
Anti alkbh5 hpa007196, by Merck Group (1 mentions)
5 protocols using goat anti mouse igg hrp zb2305
1
Inflammatory Signaling Pathway Analysis
2
Immunoblot Analysis of Protein Expression
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The immunoblot analysis was performed as described previously.15 (link) Briefly, cells were lysed with RIPA buffer (20–188, Millipore) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermo Fisher Scientific). For the separation of nuclear and cytoplasmic proteins, cells were firstly lysed with cytoplasmic lysis buffer (Tris 10 mM, NaCl 10 mM, MgCl2 3 mM, Nonidet P-40 0.1%) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermo Fisher Scientific) for 3 min, and the supernatant was collected for the detection of cytoplasmic proteins. After three washes with cytoplasmic lysis buffer, the nuclei were lysed with RIPA buffer. Protein concentrations were measured with BCA protein assay kit (Thermo Fisher Scientific). ALKBH5 (HPA007196) and METTL14 (HPA038002) antibodies were from Sigma. YTHDF1 (17479-1-AP) and PTGER4 (66921-1-Ig) antibodies were from Proteintech. METTL3 (ab195352), FTO (ab92821), and Tenascin C (ab108930) antibodies were from Abcam. WNK1 (MA5-35466) and NLRP12 (PA5-89879) antibodies were from Invitrogen. GAPDH (M171-3) and YTHDF2 (RN123PW) antibodies were from MBL. Lamin A/C (4777 S) antibody was from CST. Goat anti-rabbit IgG-HRP (ZB-2301) and goat anti-mouse IgG-HRP (ZB-2305) antibodies were from ZSGB-BIO.
3
Cellular Protein Quantification and Western Blotting
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These analyses were performed as described previously [37 (link)]. Briefly, cells were lysed with RIPA buffer (20-188; Millipore) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Then, protein concentrations were measured with a BCA protein assay kit (Thermo Fisher Scientific). Anti-ALKBH5 (HPA007196; Sigma), anti-p-STAT3 (9145T; CST), anti-STAT3 (9139T; CST), anti-G-CSFR (ab156878; Abcam), anti-β-Actin (M117-3; MBL), anti-GAPDH (M171-3; MBL), goat anti-rabbit IgG-HRP (ZB-2301; ZSGB-BIO), and goat anti-mouse IgG-HRP (ZB-2305; ZSGB-BIO) antibodies were used according to the manufacturer’s instructions.
4
Western Blot Analysis of Protein Expression
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Protein expression was analyzed using Western blotting as previously described (Yu et al. 2019 (link); Gong, Yan, et al. 2019 (link)). For Western blot analysis, primary antibodies against IL-6 (mouse monoclonal antibody, ab9324) and nephrin (rabbit monoclonal antibody, ab216341) were purchased from Abcam (UK), Anti-TGF-β1 (mouse monoclonal antibody, sc-130348) was purchased from Santa Cruz (USA), anti-podocin (rabbit polyclonal antibody, TA351459) was purchased from Origene (USA), anti-caspase-3 (rabbit polyclonal antibody, 9662S) was purchased from CST (USA), and anti-ALB (rabbit polyclonal antibody, 16475-1-AP) was purchased from Proteintech (USA). Goat anti-mouse IgG-HRP (ZB2305) and goat anti-rabbit (ZB2301) IgG-HRP secondary antibodies were purchased from ZSGB-Bio (Beijing, China). The targeted proteins were visualized with the Super Signal West Femto Chemiluminescent Substrate (Thermo Scientific Pierce), and the intensities of the visualized bands were analyzed using Quantity One software (Bio-Rad). β-Actin (mouse monoclonal antibody, TA-09, from ZSGB-Bio in Beijing, China) was used as an internal control. The data are expressed as the ratio to β-Actin.
5
Investigating Nigericin and Cisplatin Effects
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Nigericin and cisplatin were obtained from Sigma-Aldrich; Merck KGaA. Nigericin (25 mM stock) was dissolved in DMSO. cisplatin (1 mM stock) was dissolved in normal saline (0.9% NaCl). Both stock solutions were stored as aliquots at −20°C. DMSO served as a vehicle control. The following primary antibodies were used at the dilution recommended by the manufacturer (1:1,000) and purchased from Cell Signaling Technology, Inc.: Rabbit anti-Axin-2 (cat. no. 2151), rabbit anti-MMP7 (cat. no. 71031), rabbit anti-vimentin (cat. no. 5741), rabbit anti-tubulin (cat. no. 2148), rabbit anti-β-catenin (cat. no. 25362), rabbit anti-slug (cat. no. 9585), rabbit anti-E-cadherin (cat. no. 3195) and rabbit anti-small ubiquitin-like modifier 1 (SUMO1; cat. no. 4930). Mouse anti-GSK3β (cat. no. sc-377213) was used at a 1:200 dilution, and purchased from Santa Cruz Biotechnology, Inc. Goat anti-mouse IgG (HRP) (ZB-2305) and Goad anti-rabbit IgG (HRP) (ZB-2301) were used at a dilution of 1:2,500, and were purchased from ZSGB-BIO, Inc. (http://www.zsbio.com/ ).
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