In the present study, mouse monoclonal anti-MUC2 (ab11197, 1/500 WB and 1/100 IF, Abcam), rabbit monoclonal anti-TFF3 (ab108599, 1/1500 WB, Abcam), mouse monoclonal anti-SI ([53]; HSI-4/34 or Caco-3/73, 1/100 WB), rabbit monoclonal anti-DPPIV or CD26 [EPR20819] (ab215711, 1/2000 WB, Abcam), rabbit monoclonal anti-YAP/TAZ (D24E4, 1/1500 WB, Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-YAP (D8H1X, 1/1000 WB, 1/150 IF, Cell Signaling Technology), mouse monoclonal anti-TAZ (M2-616, 1/300 IF, BD Biosciences, NJ, USA) mouse monoclonal anti-CDX2-CD88 (MU392A-UC, 1/700, BioGenex, Freemont, CA, USA), mouse monoclonal anti-HNF1α [F-7] (sc-393925, 1/300 WB, Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal anti-HNF4α [C-19] (sc-6556, 1/600 WB, Santa Cruz Biotechnology) and anti-β-actin (MAB1501, 1/20,000, Millipore, Etobicoke, ON, Canada) were used as primary antibodies. In addition, AlexaFluor 488 or 594 goat anti-mouse (A11017, A11072, 1/400; Thermo Fisher Scientific, Ottawa, ON, Canada) and goat anti-rabbit (A11070, A11072, 1/400), ECL HRP-linked anti-mouse (NA931V, 1/4000, GE Healthcare, Mississauga, ON, Canada) and anti-rabbit (NA934V, 1/4000) and HRP-linked bovine anti-goat (sc-2350, 1/4000, Santa Cruz Biotechnology) were used as secondary antibodies.
Sc 2350
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Sc-2350 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is a multi-purpose device designed for various scientific applications. The core function of the Sc-2350 is to perform specific tasks required in a laboratory setting, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Sc 2370, by Santa Cruz Biotechnology (3 mentions)
Sc 2005, by Santa Cruz Biotechnology (2 mentions)
Na931v, by GE Healthcare (1 mentions)
Mab1501, by Merck Group (1 mentions)
A11017, by Thermo Fisher Scientific (1 mentions)
M2 616, by BD (1 mentions)
Ab215711, by Abcam (1 mentions)
Ab11197, by Abcam (1 mentions)
Ab108599, by Abcam (1 mentions)
Sc 393925, by Santa Cruz Biotechnology (1 mentions)
A11070, by GE Healthcare (1 mentions)
A11072, by GE Healthcare (1 mentions)
Na934v, by Santa Cruz Biotechnology (1 mentions)
Sc 6556, by Santa Cruz Biotechnology (1 mentions)
D8h1x, by Cell Signaling Technology (1 mentions)
Mu392a uc, by BioGenex (1 mentions)
Enhanced chemiluminescence western blot detection reagent, by GE Healthcare (1 mentions)
Immobilon, by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
7 protocols using sc 2350
1
Immunoblotting and Immunofluorescence Assays
2
Western Blot Analysis of Apoptosis Markers
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After treatment, the cells were harvested and lysed with radioimmunoprecipitation assay buffer (Cell Signaling Technology, Inc., Boston, MA, USA) supplemented with cocktail (Roche, Penzberg, Germany). Protein concentration was determined using a BCA Protein Assay kit (cat. no. 23225, Thermo Fisher Scientific, Inc.) and about 40 μg protein were separated with 10% SDS-PAGE by electrophoresis and were transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon; EMD Millipore, Billerica, MA, USA) using trans-blotting apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Non-fat milk (5%, w/v) dissolved in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) was used to block the PVDF membranes. The membranes were then incubated with the following primary antibodies: Procaspase-3 (1:200), procaspase-9 (1:200), cytochrome c (1:200), Bcl-2 (1:200), Bax (1:200) and β-actin (Santa Cruz Biotechnology, Inc.) overnight at 4°C. After washing with TBS-T three times, the membranes were incubated with the appropriate secondary antibodies (1:1,000; sc-2350, sc-2005 and sc-2370; Santa Cruz Biotechnology, Inc.). Finally, the protein bands were developed using enhanced chemiluminescence western blot detection reagents (GE Healthcare, Chicago, IL, USA) and were analyzed using ImageJ software 1.51s (National Institutes of Health, Bethesda, MD, USA).
3
Quantitative Immunoblotting of RELM-α in Mouse Lungs
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The mouse lung tissues were homogenized in ice-cold RIPA buffer (Cell Signaling) containing complete protease inhibitor cocktail (Roche Diagnostics GmbH) using TissueLyser LT (QIAGEN). The protein concentration in the lysate was determined using the PierceTM BCA Protein Assay Kit (Thermo Scientific). After being heated at 70 °C for 10 min in a tricine sample buffer with β-Mercaptoethanol, the proteins were separated by CriterionTM tris-tricine/peptide gel electrophoresis. And then, the proteins were transferred to PVDF membrane by Trans-Blot TurboTM Blotting system (Bio-Rad). The membranes were incubated with primary antibodies, such as a rabbit anti-murine RELM-α (500-P214, PeproTech, 1/400) or goat β-actin polyclonal antibody (sc-1616, Santa Cruz Biotechnology, 1/1000) at 4 °C overnight. After washing, the membranes were incubated with anti-rabbit (#7074, Cell Signaling, 1/1000) or anti-goat (sc-2350, Santa Cruz Biotechnology, 1/5000) HRP-conjugated secondary antibodies. The signal was visualized by SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific). The images were quantified using ImageLab software (Bio-Rad). Results are presented as mean ± SEM. The difference between the two strains C57BL/6J and C57L/J was analyzed by student t-test. P < 0.05 was considered significant.
4
Detailed Antibody Validation Protocol
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The primary antibodies used for immunofluorescence (IF) and WB analyses were rabbit monoclonal anti-ALK (WB, 1:500–1000; IF, 1:200; #3333S, Cell Signaling Technology, Danvers, MA, USA), anti-pALK Y1507 (WB, 1:1000; #14678, Cell Signaling Technology) and anti-c-Met (WB, 1:1000; #8198, Cell Signaling Technology); rat monoclonal anti-α-tubulin (WB, 1:1000; IF, 1:800; MCA78G, Bio-Rad Laboratories, Hercules, CA, USA) and anti-HA (WB, 1:1000; IF, 1:400; 3F10, Roche, Basel, Switzerland); goat anti-Lamin B (WB, 1:200–500; sc-6216, Santa Cruz Biotechnology, Dallas, TX, USA); and mouse monoclonal anti-Actin (WB, 1:2000; A3853, MilliporeSigma, Burlington, MA, USA). Secondary antibodies included Alexa Fluor 555-labeled goat anti-rat IgG (1:1000; Life Technologies, Waltham, MA, USA); 488-labeled donkey anti-rabbit and anti-rat IgG (1:800; Life Technologies) for IF. The antibodies used for WB included horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (1:4000; 711-035-152), donkey anti-rat IgG (1:4000; 712-035-153), donkey anti-mouse IgG (1:4000; 715-305-151) from Jackson Immuno Research (West Grove, PA, USA), and bovine anti-goat (1:4000; sc-2350) from Santa Cruz Biotechnology.
5
Protein Expression Analysis in Cell Lines
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The following primary antibodies were obtained from Santa Cruz Biotechnology Inc., USA; GR(H-300): sc-8992, PR(C-20) (which detects PRA and B isoforms): sc-539, AR(441): sc-7305, GAPDH(0411): sc-47724, MR(MCR, H300): sc-11412, ERα(MC-20): sc-542. The flotillin-1 (610820) antibody was purchased from BD Transduction Laboratories (USA). The following secondary antibodies were obtained from Santa Cruz Biotechnology Inc., USA; anti-mouse: sc-2005, anti-goat: sc-2350 (used as IgG for the ChIP assay) and anti-rabbit: sc-2313. The ligands dexamethasone (DEX), MPA, P4, NET-A, NET, aldosterone (ALD) and mibolerone (MIB) were obtained from Sigma-Aldrich (South Africa). Human tumour necrosis factor α (TNFα) was obtained from Celtic Diagnostics (South Africa). Protease inhibitor cocktail tablets (EDTA-free) (cat #04693159001) were obtained from Roche (South Africa). Cycloheximide (CHX) was purchased from Sigma-Aldrich (South Africa).
6
Western Blot Protein Detection Protocol
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Tissue homogenate or cultured cells were lysed with a hypotonic buffer containing 2% Nonidet-P and a protease inhibitor cocktail (Sigma-Aldrich) by sonication, which was performed three times for 3 sec on ice. The supernatant obtained subsequent to centrifugation at 2,000 × g for 15 min at 4°C, was used for protein concentration determination by the Coomassie blue method and for subsequent steps. Equal amounts of protein were used for each sample and were separated by 8–15% SDS-polyacrylamide gel and blotted onto a polyvinylidene difluoride microporous membrane (Merck Millipore, Hong Kong, China). The membranes were incubated for 1 h with a 1:1,000 dilution of primary antibody, and then washed and revealed using bovine anti-goat (catalog no., sc-2350; Santa Cruz Biotechnology, Inc.) or anti-rabbit (catalog no., sc-2370; Santa Cruz Biotechnology, Inc.) secondary antibodies with horseradish peroxidase conjugate (dilution, 1:5,000; 1 h). The peroxidase was revealed with an Amersham ECL Western Blotting Detection kit (GE Healthcare Life Sciences, Shanghai, China). Three independent experiments were performed for each western blot analysis.
7
Western Blotting of Cortical Proteins
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For WB, total proteins from ipsilateral cortical tissues were isolated. Tissue isolation, sample preparation, SDS acrylamide gel electrophoresis, blotting onto nitrocellulose membrane and membrane blocking were done as previously described 13 . The membranes were incubated with primary antibodies (Table 1) overnight at 4ºC.
The membranes were then incubated with the appropriate HRP labeled secondary antibodies: anti-rabbit antibody (1:2000; sc-2370, Santa Cruz, USA) and anti-goat antibody (1:5000; sc-2350, Santa Cruz, USA) in TBST for 1 h at room temperature. The signal was detected by enhanced chemiluminescence (ECL, Amersham Bioscience) and exposure of an X-ray film (Kodak). All films were analyzed by densitometry using the image analysis program ImageQuant ver.5.2 (Amersham). Each blot was subsequently re-probed with rabbit anti-actin antibody (1:10000; Santa Cruz Biotechnology, USA) in TBST, overnight at +4C° that served as endogenous control. The membranes were incubated with the HRP-labeled secondary anti-rabbit antibody (1:2000; sc-2370, Santa Cruz, USA) in TBST for 1 h at room temperature. The levels of the target protein were determined relative to the appropriate control values in AL rats that were set as 100%.
The membranes were then incubated with the appropriate HRP labeled secondary antibodies: anti-rabbit antibody (1:2000; sc-2370, Santa Cruz, USA) and anti-goat antibody (1:5000; sc-2350, Santa Cruz, USA) in TBST for 1 h at room temperature. The signal was detected by enhanced chemiluminescence (ECL, Amersham Bioscience) and exposure of an X-ray film (Kodak). All films were analyzed by densitometry using the image analysis program ImageQuant ver.5.2 (Amersham). Each blot was subsequently re-probed with rabbit anti-actin antibody (1:10000; Santa Cruz Biotechnology, USA) in TBST, overnight at +4C° that served as endogenous control. The membranes were incubated with the HRP-labeled secondary anti-rabbit antibody (1:2000; sc-2370, Santa Cruz, USA) in TBST for 1 h at room temperature. The levels of the target protein were determined relative to the appropriate control values in AL rats that were set as 100%.
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