The protein of treated ATDC5 cells was lysed using lysis buffer (Promega) supplemented with protease inhibitors (Roche, Mannheim, Germany). Each protein sample was quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). Then, proteins were separated by SDS-PAGE and were blotted onto polyvinylidene difluoride membranes, followed by blocking with 5% non-fat milk. The blocked membranes were respectively incubated with anti-Bcl-2-associated X protein (Bax, ab182733), anti-B cell lymphoma-2 (Bcl-2, ab196495), anti-pro caspase-3 (ab90437), anti-cleaved caspase-3 (ab49822), anti-p65 (ab16502), anti-phosphorylated p65 (p-p65, ab86299), anti-inhibitor of nuclear factor κB α (IκBα, ab32518), anti-phosphorylated IκBα (p-IκBα, ab133462), anti-Notch1 (ab52627), anti-Notch2 (ab137665), anti-Notch3 (ab23426), anti-GAPDH (ab181603) (all Abcam, Cambridge, UK), anti-pro caspase-9 (9508), anti-cleaved caspase-9 (9509) (both Cell Signaling Technology, Beverly, MA, USA), or anti-PDCD4 (sc-376430; Santa Cruz, Santa Cruz, CA, USA) at 4 °C overnight. Then, after rinsing thrice, membranes were incubated with secondary antibodies marked by horseradish peroxidase for 1 hr at room temperature. After rinsing again, the bands on the membranes were visualized by ECL detection systems (Sigma-Aldrich).
Ab90437
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab90437 is a primary antibody that binds to the target protein. It is suitable for use in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ab49822, by Abcam (2 mentions)
Ab16502, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab23426, by Abcam (1 mentions)
Ab137665, by Abcam (1 mentions)
Ab52627, by Abcam (1 mentions)
Lysis buffer, by Promega (1 mentions)
Ab182733, by Abcam (1 mentions)
Ecl detection system, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Ab2426, by Abcam (1 mentions)
Sc5276, by Santa Cruz Biotechnology (1 mentions)
Mannitol, by Merck Group (1 mentions)
Ab2413, by Abcam (1 mentions)
Bortezomib, by Merck Group (1 mentions)
Ab25758, by Abcam (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Emodin, by Merck Group (1 mentions)
N benzyloxycarbonyl val ala asp fluoromethylketone zvad fmk, by Merck Group (1 mentions)
15 protocols using ab90437
1
Western Blot Analysis of Apoptosis Markers
2
Investigating Apoptosis Pathways in Cells
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Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) was purchased form Gibco (Life technologies, Shanghai, China). An antibody that recognizes both the long and short isoforms of cFLIP (cFLIPL and cFLIPS) (sc-5276) was purchased from santa cruz (Santa Cruz, USA). Another one against caspase-8 was from Alexis Biochemicals. Other antibodies against caspase-3 (ab90437), caspase-9 (ab25758), PCNA (ab2426) and fibronectin (ab2413) were from abcam (Cambridge, UK). Emodin, streptozotocin (STZ), D-glucose, mannitol, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk), bortezomib and other chemicals were purchased from Sigma (St. Louis, USA) unless otherwise stated.
3
Protein Expression Analysis of Apoptosis and UPR Pathways
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The total protein was extracted by the modified RIPA buffer (P0013B; Beyotime, Shanghai, China), and the concentration was quantitated by the BCA protein assay kit (P0010; Beyotime, Shanghai, China). Then, the total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, and then the membranes were blocked in 5% skim milk diluted with TBST for 1 h at room temperature and probed with primary antibodies, including MFG-E8 (R&D Systems, AF2805, 1 : 500), BCL2 (Abcam, ab117115, 1 : 500), BAX (Abcam, ab216494, 1 : 500), cleaved caspase-3 (Abcam, ab90437, 1 : 500), GRP-78 (Abcam, ab230508, 1 : 500), XBP-1 (Abcam, ab230508, 1 : 500), ATF-6 (Abcam, ab203119, 1 : 500), ATF-4 (Abcam, ab23760, 1 : 500), CHOP (Abcam, ab23760, 1 : 500), p-PI3K (Abcam, ab125633, 1 : 500), PI3K (Abcam, ab154583, 1 : 500), p-AKT (Abcam, ab18785, 1 : 1000), AKT (Abcam, ab28422, 1 : 500), and β-actin (Abcam, ab209857, 1 : 1000) at 4°C overnight. Then, the membranes were incubated with a secondary rabbit anti-rabbit antibody (1 : 1000) the next day at room temperature for 1 h. The ImageJ software was employed for quantitation of the immunoreactive bands.
4
Western Blot Analysis of Signaling Proteins
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Western blotting was performed as previously described30 . Cells were lysed in RIPA buffer (Thermo Fisher) with protease inhibitor cocktail (Roche). The supernatant of the lysate was collected and denatured with the same volume of SDS sample buffer (0.5 M Tris-HCl, pH 6.8; 10% SDS, 50% glycerin, 2-mercaptoethanol). The protein samples were resolved viaSDS-PAGE and blotted onto PVDF membranes (Bio-Rad). PVDF membrane was cut to enable blotting for multiple antibodies simultaneously which were then labeled with the primary antibody overnight at 4 °C. Anti-HIP antibody (Abcam, Ab39208), Anti-Gli1 antibody (Biolegend, 642401), Anti-VEGF antibody (Biolegend, 512901), and Anti-Caspase-3 antibody (Abcam, Ab90437) at a dilution of 1:1000 were used. Then secondary antibody was incubated for 1 h at RT and detected using ECL Plus reagents(Thermo Fisher).
5
Western Blot Analysis of Cell Apoptosis
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Cell lysates were prepared, and the protein concentrations were quantified with a BCA protein assay kit (CWBIO, CW2011S, Beijing, China). Samples with equal amounts of proteins were separated by 12% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk at room temperature for 1 h, the membranes were incubated at 4°C overnight with primary antibodies (1:1000) against cleaved caspase-3 (Cell Signaling Technology, Aps175, Boston, USA), caspase-3 (Abcam, ab90437, Cambridge, UK), LC3 (Abcam, ab128025, Cambridge, UK), Beclin1 (BD, 612113, New York, USA), p62 (Abcam, ab56416, Cambridge, UK) or β-actin (1:5000; CWBIO, CW0096M, Beijing, China). The next day, the membranes were washed with TBST three times and incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1:5000, CWBIO, Beijing, China) at room temperature for 1 h. The blots were visualized with a cECL Western blot kit (CWBIO, CW0049M, Beijing, China), and the images were analyzed by ImageJ.
6
Melatonin Effects on Granulosa Cells
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The antibodies including CASP3 rabbit polyclonal antibody (ab90437) and anti-MTNR1B rabbit polyclonal antibody (ab203346) were purchased from Abcam, Cambridge, MA, USA. All the other antibodies including MTNR1A goat monoclonal antibody (SC-13186), BAX rabbit polyclonal antibody (SC-526), TP53 mouse monoclonal antibody (SC-99), BCL2 rabbit polyclonal antibody (SC-492), ACTB (actin beta) mouse monoclonal antibody (SC-47778), goat anti-rabbit lgG-HRP(SC-2054), chicken anti-goat lgG-HRP (SC-2961), and goat anti-mouse lgG-HRP (SC-2005) were purchased from the Santa Cruz Biotechnology, Inc., Dallas, TX, USA.
Melatonin was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in ethanol at the concentration of 100 μg/ml, and then diluted to the final concentration (1,200 pg/ml) with Dulbecco’s modified essential medium (DMEM) (Gibco, Grand Island, NY, USA) before adding to the cultured GCs.
Melatonin was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in ethanol at the concentration of 100 μg/ml, and then diluted to the final concentration (1,200 pg/ml) with Dulbecco’s modified essential medium (DMEM) (Gibco, Grand Island, NY, USA) before adding to the cultured GCs.
7
Comprehensive Protein Expression Analysis
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For Western Blotting cells of at least three independent biological replicates were treated, as indicated, and lysed with 1xRIPA buffer; Western Blotting and detection of the proteins with specific antibodies was performed as previously described elsewhere and according to standard protocols (REF). Antibodies against Nestin (2393925), CD133 (2438662) and Klf4 (2116008) were purchased from Millipore (Darmstadt, Germany), against alpha-Tubulin (DLN-09992) from Dianova (Hamburg, Germany) and Oct4 (0421) from DCS (Berlin, Germany). Antibodies against cleaved Caspase-3 (ab90437) and GAPDH (ab8245) were purchased from abcam (Cambridge, UK), the anti β-Actin-Peroxidase antibody (AC-15) was purchased from Sigma-Aldrich (Darmstadt, Germany). The self-made antibody detecting Δ-catenin was previously described in [18 (link)].
8
Western Blot Analysis of Apoptosis Markers
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Cells were lysed in radioimmunoprecipitation assay buffer (Beyotime, Nantong, China) containing protease inhibitors (Sigma). Normalized protein samples (50 μg/lane) were resolved by SDS/PAGE and transferred on to nitrocellulose membranes. The following primary antibodies were used for the present study: anti-RB1 monoclonal antibody (Abcam, ab24, Cambridge, MA, U.S.A.), rabbit anti-caspase-3 polyclonal antibody (ab90437, Abcam), rabbit anti-poly (ADP-ribose) polymerase (PARP) polyclonal antibody (ab194586, Abcam), mouse anti-Bcl-2 monoclonal antibody (#15071, Cell Signaling Technology, Danvers, MA, U.S.A.), rabbit anti-Bax polyclonal antibody (#2772, Cell Signaling Technology), and anti-GAPDH monoclonal antibody (Beyotime). HRP–conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Protein bands were visualized with the chemiluminescent system (Cell Signaling Technology). Densitometry was performed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, U.S.A.).
9
Protein Expression Analysis Protocol
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Cells were lysed using lysis buffer solution (Beyotime); then, proteins were quantified using the BCA method. Protein extracts (50 μg) were separated by 8% SDS-PAGE, then transferred onto polyvinylidene difluoride membranes. Next, proteins were probed with primary antibodies against mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2, 1 μg/mL; Abcam, ab55271), Bax (1 : 500; Abcam, ab53154), Bcl-2 (1 μg/mL; Abcam, ab185002), Caspase3 (1 : 5000; Abcam, ab90437), MTF1 (1 : 1000; Abcam, ab236401), nuclear factor E2-related factor 2 (Nrf2, 1 : 1000; Abcam, ab89443), Lamin B (1 : 4000; Abcam, ab151735), and β-actin (1 : 500; Abcam, ab179467). After washing, membranes were incubated with secondary goat anti-mouse IgG (1 : 1000, EMD Millipore, Billerica, MA, USA) antibody. Band intensity was quantified using ImageJ 1.52 software (National Institutes of Health, Bethesda, MD, USA).
10
Immunohistochemical Analysis of Apoptosis and Proliferation
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The cleaved caspase‐3 (ab90437; 1:1000; Abcam) and Ki67 (ab92742; 1:1000; Abcam) contents in tumor were distinguished by IHC assay. The detailed procedures were conducted as reported by Zou et al.28 Briefly, tumor tissues from mice were cut into 4‐μm thickness slides, followed by dewaxing, rehydration, and antigen retrieval. Then, the slides were challenged with the antibodies targeting cleaved caspase‐3 and Ki67. After incubation of matched secondary antibody, the slides were stained using the DAB kit from Abcam. The positive staining was observed by light microscopy.
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