Clarified cell lysates were resolved by SDS-PAGE, blotted onto nitrocellulose membranes and immunoprobed with the relevant primary and secondary antibody combinations. Anti-pTrkB Tyr516 (4619), anti-pTrkB Tyr706/707 (4621), anti-pTrkB, Tyr816 (4168), anti-pERK1/2 (4051), anti-pAKT (4051), anti-TrkB (4603), anti-AKT (4685) and anti-ERK1/2 (4695) were sourced from Cell Signalling Technologies. Anti-GAPDH (IMG-5143A) was obtained from Imgenex; Goat anti-rabbit HRP conjugate (PO448) and Goat anti-mouse HRP conjugate (PO447) were sourced from Dako. Goat anti-mouse 800CW conjugate and Goat anti-rabbit 680 conjugate were sourced from LI-COR. Immunoreactive signals were detected using enhanced chemiluminescence reagent (Amersham) or using the Odyssey infra-red reader.
Anti erk1 2 4695
Manufactured by Cell Signaling Technology
Sourced in United States, China
Anti-ERK1/2 #4695 is a laboratory reagent used for the detection and quantification of Extracellular Signal-Regulated Kinase 1 and 2 (ERK1/2) proteins. It is a primary antibody that specifically binds to these proteins, allowing for their identification and measurement in various experimental settings.
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Lab products found in correlation
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Complete edta free, by Roche (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Promega (1 mentions)
Anti v5 r960 25, by Thermo Fisher Scientific (1 mentions)
Anti gapdh 5174, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3 9664, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2 4370, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Anti hif 1α 36169, by Cell Signaling Technology (1 mentions)
U0126, by Merck Group (1 mentions)
Ab7817, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
Ab7778, by Abcam (1 mentions)
Recombinant tgf β1, by Merck Group (1 mentions)
5 protocols using anti erk1 2 4695
1
Western Blot Analysis of Phosphorylated TrkB
2
Western Blot Analysis of Cellular Proteins
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For Western blot analysis, total protein extract was isolated from A172 and LN229 cells by lysis in NP-40 Buffer containing protease and phosphatase inhibitors (complete EDTA-free; Roche Applied Science, Monza, Italy), and protein concentrations were determined by Bradford method. Equivalent amounts of protein extract were separated by electrophoresis on 10% or 12% SDS-PAGE gels and blotted onto nitrocellulose. The membranes were blocked with 5% non-fat dry milk and 0.1% Tween-20 in Phosphate-buffered saline and then incubated with antibodies followed by appropriate horseradish peroxidase-conjugated secondary antibodies (Promega, Milan, Italy). The primary antibodies employed for protein detection were: anti-CXCL14 #MAB866, R&D Systems (1:500); anti-V5 #R960-25, Invitrogen (1:5000); anti-β-actin #A2066, Sigma (1:10,000); anti-ERK1/2 #4695, Cell Signaling (1:1000) and anti-phospho ERK1/2 #9106, Cell Signaling (1:2000).
3
Western Blot Analysis of Protein Targets
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Following cells lysis, protein cell extracts (40 µg) were separated on 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoblots were performed overnight using the following primary antibodies diluted 1:1,000: anti-HIF-1α #36169, anti-phospho-ERK1/2 #4370, anti-ERK1/2 #4695, anti-cleaved caspase-3 #9664S and anti-GAPDH #5174S (Cell Signaling Technology, Danvers, USA), anti-VEGF-A #46154 (Abcam, Cambridge, UK). An Ig anti-rabbit #7074S 1:1000 secondary antibody was used (Cell Signaling Technology, Danvers, USA). Blots were then developed using ECL (Merck, Milan, Italy) and images were acquired by Image Quant Las 4000mini (GE Healthcare Life Sciences, Milan, Italy).
4
Fibroblast Growth Factor Signaling Assay
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In this study, anti‐alpha smooth muscle actin (ab7817), anti‐discoidin domain receptor 2 (DDR2) antibody (ab221812), anti‐collagen type I antibody (ab34710), and anti‐collagen type III antibody (ab7778) were purchased from Abcam company (Shanghai, China), and anti‐ERK1/2 (#4695) and anti‐p‐ERK1/2 (#5726) were purchased from Cell Signaling Technology (Danvers, MA, USA). Recombinant TGF‐β1 and U0126 were ordered from Sigma‐Aldrich (St. Louis, MO, USA). Recombinant FGF2 (18 kDa) with a hexa‐histidine tag was prepared as described [17 , 18 (link)]. The purified FGF2 was diluted to 20 μg·mL−1 with PBS for further experiments.
5
Generation and Validation of Anti-pSer483 RIOK2 Antibodies
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Anti-phospho Ser483 RIOK2 antibodies were generated according to immunization protocol from Covalab. Briefly, a peptide phosphorylated at Ser483 (MNQYRTRTL(Sp)ITS) conjugated to a carrier protein was first used to immunize rabbits. Following the final bleed, the immune serum was loaded onto a column with the control peptide (MNQYRTRTLSITS) coupled to agarose beads, thus retaining unmodified peptide-specific antibodies. The flow-through was then loaded onto a column with the modified peptide coupled to agarose beads, thus retaining modified peptide-specific antibodies. After elution, the eluate was assayed by ELISA against both peptides to control its immuno-reactivity and its specificity against the modification. Anti-phospho-ERK 1/2 (T202/Y204, #4370), anti-phospho-RSK (S380), anti-phospho-(Ser/Thr) Akt Substrate (#9614) and anti-ERK1/2 (#4695) antibodies were purchased from Cell Signaling Technology. Anti-RSK1(#GTX111050), anti-RSK2 (#38–6800), anti-puromycin (clone 12D10, #MABE343) and anti-HA (clone 16B12, #MMS-101R-500) antibodies were purchased from Genetex, Life Technologies, Millipore and Covance, respectively. Anti-RIOK2 (#A302-450A) and anti-ENP1 (#A304-568A) antibodies were purchased from Bethyl Laboratories.
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