Sc 292838

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Sc-292838 is a laboratory equipment product. It is designed for use in scientific research and experimentation. The core function of Sc-292838 is to provide a controlled environment for various experimental procedures. No further details about its intended use or performance specifications are available.

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Lab products found in correlation

9 protocols using sc 292838

Western Blot Analysis of Protein Targets

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Equal amounts of protein were separated by a reducing SDS-PAGE and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane [31 (link), 32 (link)]. After incubation in blocking buffer (5% nonfat dry milk, TBS and 0.05% Tween-20) at room temperature for 2 h, the membranes were incubated with primary antibodies overnight at 4°C. The antibodies included rabbit polyclonal anti-CTGF antibody (1:400, BA0752, Boster Biotechnology), rabbit polyclonal anti-SR-1B antibody (1:500, BS2765, Bioworld Technology, Inc.), rabbit polyclonal anti-ERK1/2 antibody (1:500, sc-292838, Santa Cruz Biotechnology), mouse monoclonal anti-p-ERK1/2 (1:500, sc-81492, Santa Cruz Biotechnology), rabbit polyclonal anti-bcl-2 antibody (1:500, BS1511, Bioworld Technology, Inc.), rabbit polyclonal anti-bcl-xl antibody (1:500, BS1032, Bioworld Technology, Inc.), rabbit polyclonal anti-caspase-3 antibody (1:400, BS61583, Bioworld Technology, Inc.) and mouse polyclonal anti-β-actin antibody (1:2000, sc-47778, Santa Cruz Biotechnology]. The immunoreactive bands were visualized using the corresponding horseradish peroxidase-conjugated secondary antibodies and super ECL plus (Thermo Fisher Scientific, Waltham, MA, USA). The relative protein expression was quantified by densitometry using Quantity One (Bio-Rad Laboratories, California, USA).

Bai Y., Li Z.X., Zhao Y.T., Liu M., Wang Y., Lian G.C., Zhao Q, & Wang H.L. (2017). PCPA protects against monocrotaline-induced pulmonary arterial remodeling in rats: potential roles of connective tissue growth factor. Oncotarget, 8(67), 111642-111655.

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Signaling Pathways in EL4 Cells

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EL4 cells transfected overnight with pEGFP-C3-siOCILRP2 were stimulated with anti-CD3/CD28 antibodies. For the other two groups, EL4 cells were stimulated with or without anti-CD3/CD28 antibodies. Cell lysates were prepared in SDS sample buffer (Tris-HCl, SDS, and 20% glycerol) and detected using SDS-PAGE. The proteins were electrotransferred onto Immobilon-P PVDF membranes (0.45 µm, Millipore, USA) and probed with the appropriate primary antibodies against the following: p-Raf-1 (Ser 338) (sc-12358, Santa Cruz, 1∶200), caspase-8 p18 (G-1) (sc-166596, Santa Cruz, 1∶500), caspase-3 p17 (B-4) (sc-271028, Santa Cruz, 1∶500), JNK1 (D-6) (sc-137018, Santa Cruz, 1∶500), p-JNK1/2/3 (T183+Y185) pAb (BS4322, Bioworld Technology, 1∶500), ERK 1/2 (H-72) (sc-292838, Santa Cruz, 1∶200), p-ERK 1/2 (Thr202/Tyr204) (sc-16982, Santa Cruz, 1∶200), IκB-alpha (N-terminus) mAb (MB0106, Bioworld Technology, 1∶1000), β-actin (Sigma, 1∶5,000), and β-tubulin (Sigma, 1∶5000)). After incubation with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz, 1∶10,000) for 3-4 h, the membranes were washed with PBST. Immunoreactivity was visualized using an ECL system (Perkin Elmer, USA), and densitometry scanning of the intensity of the bands was quantified using ImageJ.

Lou Q., Zhang W., Liu G, & Ma Y. (2014). The C-Type Lectin OCILRP2 Costimulates EL4 T Cell Activation via the DAP12-Raf-MAP Kinase Pathway. PLoS ONE, 9(11), e113218.

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Immunoblot Analysis of Cell Signaling

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Western blotting analysis was performed using standard techniques. The following antibodies were used: LSP1 (ab133506, Abcam), ERK1/2 (sc‐292838, Santa Cruz Biotechnology, Dallas, TX, USA), phosphorylated ERK1/2 (p‐ERK1/2; sc‐16982, Santa Cruz Biotechnology), cyclin D1 (ab137875, Abcam), B‐cell lymphoma 2 (Bcl2; ab32124, Abcam) and β‐actin (sc‐4778, Santa Cruz Biotechnology).

Zhang H., Wang Y., Liu Z., Yao B., Dou C., Xu M., Li Q., Jia Y., Wu S., Tu K, & Liu Q. (2016). Lymphocyte‐specific protein 1 inhibits the growth of hepatocellular carcinoma by suppressing ERK1/2 phosphorylation. FEBS Open Bio, 6(12), 1227-1237.

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Western Blot Analysis of EMT Markers

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Several target proteins were detected their expression by Western blot. Proteins were separated on a 12% SDS-PAGE gel and transferred onto a PVDF membrane at 100 V for 1 h. The specific primary antibodies, including vimentin (1:500, sc373717, Santa Cruz), E-cadherin (1:500, sc-31021, Santa Cruz), N-cadherin (1:500, sc-31031, Santa Cruz), twist (1:500, sc-81417, Santa Cruz), STC2 (1:500, sc-14350, Santa Cruz), ERK (1:500, sc-292838, Santa Cruz), MEK (1:500, sc-436, Santa Cruz), p-ERK (1:500, sc- 136521, Santa Cruz), p-MEK (1:1000, 9121, Cell signaling), Akt (1:1000, 4961, Cell signaling) and phospho Akt (1:1000, 2118–1, Epitomics), were diluted in TBST buffer (50 mM Tris–HCl, with 150 mM NaCl, 0.1% Tween-20, pH 7.4) to incubate PVDF membrane at 4°C overnight. The corresponding secondary antibodies, conjugated horseradish peroxidase, were subsequently incubated with the PVDF membrane at 37°C for 1 h. Signal detection was performed with HRP substrates (WBLUR0100, Millipore). The detection of GAPDH against its antibody (1:1000, sc- 365062, Santa Cruz) was taken as a control.
For serum STC2 detection, the high abundant serum albumin and IgG was removed by using a reagent kit (ProteoExtract Albumin/IgG Removal Kit, 122642, Calbiochem, San Diego, CA) [38 (link)]. Reversible Ponceau staining was used as a loading control.

Chen B., Zeng X., He Y., Wang X., Liang Z., Liu J., Zhang P., Zhu H., Xu N, & Liang S. (2016). STC2 promotes the epithelial-mesenchymal transition of colorectal cancer cells through AKT-ERK signaling pathways. Oncotarget, 7(44), 71400-71416.

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Quantifying ATP1A1 and MAPK Pathway in Cell Lines

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OS-RC-2 and 786-0 cells were transfected with pYR-ATP1A1 or pYR plasmids for 48 h, and cell pellets were collected. Cell pellets and grated tissues were dissolved with RIPA buffer (Cat.# P0013C, Beyotime, China) to extract proteins. 100 μg proteins were separated on 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Cat.# IPVH00010, China). After blocking with 5% skim milk (OXOID), the PVDF membrane was incubated with primary antibodies overnight at 4 °C. The primary antibodies against ATP1A1 (1:200, ab2872, Abcam, UK), MEK1/2 (1:500, sc436, Santa Cruz, USA), p-MEK1/2(1:500, sc-81503, Santa Cruz, USA), ERK (1:500, sc-292838, Santa Cruz), p-ERK (1:500, sc-136521, Santa Cruz) β-actin (1:500, sc-1616, Santa Cruz, USA), and GAPDH (1:1000, sc-365062, Santa Cruz) were respectively diluted in TBST buffer (50 mM Tris–HCl, with 150 mM NaCl, 0.1% Tween-20, pH 7.4). Then the PVDF was incubated with the HRP-tagging secondary antibodies (ZSGB-BIO, China) at 37 °C for 1 h. Signals were finally detected with Western blot reagent ECL (Amersham Biosciences).

Zhang D., Zhang P., Yang P., He Y., Wang X., Yang Y., Zhu H., Xu N, & Liang S. (2017). Downregulation of ATP1A1 promotes cancer development in renal cell carcinoma. Clinical Proteomics, 14, 15.

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Western Blot Analysis of Zyxin and Erk2

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For western blot analysis, the cells were washed twice with ice-cold PBS and lysed for 10 min in a cold radioimmunoprecipitation assay (RIPA) buffer (with protease inhibitor cocktail) on ice and then centrifuged at 14,000 rpm for 15 min. Proteins were resolved by 10% SDS-PAGE and transferred to hydrophilic polyvinylidene fluoride (PVDF) membranes, blocked with 5% nonfat milk in TBST for 1 h at room temperature, and then incubated with primary antibodies against zyxin (mouse, 1:500, H00007791-M01, RRID: AB_2221180; Abnova, Taipei, Taiwan) and Erk2 (rabbit, 1:1000, sc-292838, RRID: AB_2650548; Santa Cruz Biotechnology, Dallas, TX, United States) at 4°C overnight. The membranes were then washed four times for 10 min in TBST and then incubated with a second antibody at room temperature for 1 h. After three washes in TBST for 10 min, the membrane was assessed using an enhanced chemiluminescence western blotting detection kit.

Kang X., Deng Y., Cao Y., Huo Y, & Luo J. (2021). Zyxin Mediates Vascular Repair via Endothelial Migration Promoted by Forskolin in Mice. Frontiers in Physiology, 12, 741699.

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Immunohistochemical Profiling of Endothelial Cells

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The following chemicals, unless specifically indicated, were from Sigma-Aldrich (St. Louis, MO, United States): forskolin (F6886), epinephrine (E4642), heparin (H3149), 4', 6-diamidino-2 phenylindole dihydrochloride (DAPI, D8417), mitomycin C (M5353), and dimethylsulfoxide (D2650). Anti ERK2 (rabbit, 1:1000, sc-292838, RRID: AB_2650548; Santa Cruz Biotechnology, Dallas, TX, United States) and anti-zyxin (mouse, 1:500, H00007791-M01, RRID: AB_2221180; Abnova, Taipei, Taiwan) were used for western blot. Anti Pecam1 (rat, 1:500, 553370, RRID: AB_394816; BD Bioscience, San Jose, CA, United States) and anti Ki67 (rabbit, 1:500, ab15580, RRID: AB_443209; Abcam, Cambridge, MA, United States) were used for whole-mount staining. anti-zyxin (mouse, 1:500, H00007791-M01, RRID: AB_2221180; Abnova, Taipei, Taiwan) and anti Ki67 (rabbit, 1:500, ab15580, RRID: AB_443209; Abcam, Cambridge, United Kingdom) were used for immunohistochemistry and immunofluorescence staining.

Kang X., Deng Y., Cao Y., Huo Y, & Luo J. (2021). Zyxin Mediates Vascular Repair via Endothelial Migration Promoted by Forskolin in Mice. Frontiers in Physiology, 12, 741699.

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Western Blot Analysis of Signaling Proteins

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A western blot analysis was performed as described previously.^{11 (link)} The antibodies that were used included anti-livin (SC-30161, 1:1000). Antibodies against ERK1/2 (sc-292838, 1:1000) and phospho-ERK1/2 (sc-23759-R, 1:2000) were purchased from Santa Cruz Biotechnology (USA). Antibodies against HER2 (ab8054, 1:500) were purchased from Abcam. Anti-phospho-AKT (SC-33437, 1:2000), anti-AKT (SC-1618, 1:1000), and mouse anti-fibronectin (SC18827, 1:1000) antibodies were purchased from Santa Cruz Biotechnology. Anti-vimentin (BS1776, 1:1000), anti-E-cadherin (BS1098, 1:1000), and anti-N-cadherin (BS222, 1:1000) antibodies were purchased from Bioworld Technology, and anti-rabbit peroxidase-conjugated secondary antibodies were purchased from Promega. An inhibitor of ERK1/2 (PD98059) was obtained from Santa Cruz Biotechnology (USA) and an inhibitor of AKT (LY294002) was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Li F., Zhang L., Feng F., Zheng K., Li Y., Wang T, & Ren G. (2018). Livin participates in resistance to trastuzumab therapy for breast cancer through ERK1/2 and AKT pathways and promotes EMT-like phenotype. RSC Advances, 8(50), 28588-28601.

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Osteogenic Protein Expression under Varying Oxygen Conditions

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Protein expression of ERK1/2, p-ERK1/2 and osteogenic maker (RUNX2) was analyzed by Western blotting assay. Briefly, after total protein of rTDSCs osteogenic-cultured in different oxygen tension conditions for 21 days was extracted with RIPA solution (Beyotime, China), protein samples were subjected to SDS-PAGE and transferred to PVDF membrane (Roche). Then, the PVDF membrane was blocked with 5% bovine serum albumin (BSA) and incubated with primary antibodies (ERK1/2, 1:500, sc-292838, Santa Cruz; p-ERK1/2, 1:500, sc-101761, Santa Cruz; RUNX2, 1:500, sc-390351, Santa Cruz; β-actin, 1:1000, 60008-1-Ig, Proteintech) overnight at 4°C and HRP-conjugated secondary antibodies (ZSGB-BIO, China) for 2 hours at room temperature. Finally, protein bands on the PVDF membrane were visualized using the SuperSignal West Pico Trial Kit (Thermo) and analyzed using the Image J software (National Institutes of Health, USA).

Li P., Xu Y., Gan Y., Song L., Zhang C., Wang L, & Zhou Q. (2016). Role of the ERK1/2 Signaling Pathway in Osteogenesis of Rat Tendon-Derived Stem Cells in Normoxic and Hypoxic Cultures. International Journal of Medical Sciences, 13(8), 629-637.

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