Pdgfrb−/− MEFs or 10T1/2 fibroblasts were transduced, sorted by FACS analysis for GFP expression, and replated onto standard cell culture plates. Expression of P-WT and P-Ex12 was assessed by qRT-PCR or western blotting, essentially as previously described (Silva et al. 2005 (link)). RT-PCR primers were designed using the National Library of Medicine PRIMER-BLAST tool, and obtained from Sigma-Aldrich. For western blotting, proteins were solubilized in RIPA lysis buffer supplemented with PhosStop Phosphotase inhibitor and Roche Complete Protease inhibitor (Roche). Primary antibodies recognized human and mouse PDGFRB (EMD Millipore, #04-825), phospho-p42/44 (Cell signaling #9102), total MAPK (Cell Signaling, 9102S), and HSC-70 (Santa Cruz SC-729). Secondary antibodies included anti-goat (Jackson ImmunoResearch 705-035-003), anti-rabbit (Jackson ImmunoResearch 111-035-003), and anti-mouse (Jackson ImmunoResearch 715-035-0150, and protein expression was detected by using both CareStream film (Sigma-Aldrich Z3730398) and Bio-Rad Clarity Enhanced Chemiluminescence (Bio-Rad 1705060)
Phospho p42 44
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-p42/44 is a lab equipment product that detects phosphorylated forms of the p42/44 MAPK (ERK1/2) proteins. This product enables the monitoring and analysis of the activation state of the p42/44 MAPK pathway, which is crucial for various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Hsc70, by Santa Cruz Biotechnology (1 mentions)
Primer blast tool, by Merck Group (1 mentions)
Phosstop phosphotase inhibitor, by Roche (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Phospho src, by Cell Signaling Technology (1 mentions)
Phospho mek1 2, by Cell Signaling Technology (1 mentions)
Phospho egfr, by Cell Signaling Technology (1 mentions)
Phospho her2, by Cell Signaling Technology (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Anti mouse and anti rabbit igg, by Cytiva (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Cwr22rv1, by American Type Culture Collection (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Pwr 1e, by American Type Culture Collection (1 mentions)
Z vad fmk, by Santa Cruz Biotechnology (1 mentions)
P mdm2, by Cell Signaling Technology (1 mentions)
Ecl detecting kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using phospho p42 44
1
Measuring PDGFRB Expression and Signaling
2
ERK1/2 Activation by Extracellular Calcium
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750 thousand HEK293 cells were seeded in 6 wells plates and transfected in 6 replicates with WT or mutant vectors using Lipofectamine 2000 (Invitrogen). After 24 h cells were starved with DMEM/F12 without Mg++ and with minimal Ca++ concentration (0,5 mM final). 48 h after the transfection, cells were stimulated in triplicates for each vector with 4 or 10 mM CaCl2 for 5 min. Whole-cell extracts were made with RIPA buffer supplemented with cocktail inhibitors as above described. 80 ug of proteins were loaded onto a 12% SDS-PAGE and analyzed for expression of phosphorylated and total ERK1/2 proteins by immunoblotting with rabbit Phospho p42/44 and rabbit Total p42/44 antibodies, respectively (Cell Signaling Technology) according to the manufacturer’s protocol. Secondary antibody was as above described. The Image J-National Institutes of Health image processing program (http://rsb.info.nih.gov/ij/ ) was used for signal densitometry by determining the ratios of the phosphorylated to nonphosphorylated ERK1/2 signals at various extracellular calcium concentrations and then normalized to the ratio of phosphorylated to unphosphorylated ERK1/2 at 10 mM [Ca++].
3
Protein Expression Analysis of Human Liver Samples
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Human FL-HCCs, normal liver samples, and hepatocyte cultures were homogenized in ice-cold radioimmunoprecipitation (RIPA) buffer containing protease inhibitors, and protein concentrations were measured using the BCA Protein Assay (Pierce, Rockford, IL). Equal amounts of protein were separated by SDS-PAGE, transferred to Immobilon-P membranes (Millipore, Bedford, MA) and incubated at 4 degrees C overnight with the following primary antibodies: NTS (sc-20806, Santa Cruz), NTSR1 (sc-376958, Santa Cruz), NTSR2 (NB100-56472, Novus Biologicals), PCSK1 (sc-100578, Santa Cruz), phospho-p70 S6 Kinase (Thr389, #97596), phospho-p42/44 (#4370), phospho-MEK1/2 (Ser217/221, #9154), phospho-Src (Tyr416, #2101), phospho-EGFR (Tyr1068, #3777), phospho-HER2 (Tyr1248, #2247; all Cell Signaling) and β-Actin (A5441, Sigma, St. Louis, MO).
4
Quantification of Signaling Protein Levels
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The protein extracts were prepared as described previously [14 (link)]. Following SDS-PAGE and electroblotting, the membranes were incubated with the following primary antibodies: p65, phospho-p65 (Ser536), IκBα p38, p42/44, JNK, pospho-p38, phospho-p42/44, phospho-JNK, Phospho-Akt, Akt and tubulin (Cell Signaling, Danvers, MA, USA); Laminin A/B (Santa Cruz, Dallas, Texas, USA); SP1 (Sigma-Aldrich, St. Louis, MO, USA). Primary antibody application was followed by incubation with horseradish peroxidase-conjugated secondary antibodies (anti-mouse and anti-rabbit IgG, Amersham, Uppsala, Sweden; anti-goat, Dako, Glostrup, Denmark). The blots were visualized using an enhanced chemiluminescence detection system (ECL) (Amersham, Freiburg, Germany) according to the manufacturer’s instructions. Densitometry was used to quantify band intensities using ImageJ (v1.29 s). Optical densities of the bands were corrected for loading differences based on corresponding control bands.
5
Prostate Cancer Cell Line Characterization
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CWR22Rv1, LNCaP and DU145 cells, and immortalized untransformed prostate epithelial cells (PWR-1E) were purchased from ATCC. PI3k inhibitor (LY294002), MAPK inhibitor (U0126), rabbit polyclonal antibodies against AR, pMdm2, β-actin, Gapdh, pAkt (S473), pAkt (T308), CHIP, PARP, caspase 3, pMdm2, p42/44, phospho p42/44, secondary antibodies, anti-mouse and anti-rabbit HRP were purchased from Cell Signaling. Cell culture reagents (FBS, RPMI, and DMEM) were from Invitrogen. Gal and VNPT55 were synthesized in our laboratory as previously reported [21 , 25 (link)]. PhosphoAR (pAR) was purchased from Imgenex. AR-V7 (AR3) expression plasmid was obtained from Dr. Yun Qiu, University of Maryland, Baltimore. AR-V7 antibody was purchased from Precision Antibodies. Dr. Stephen Plymate, University of Washington School of Medicine, Seattle donated ARv567es expression plasmid. Mdm2 Monoclonal antibody and polyclonal antibodies against AR N20, ERβ, PR, RARα, RARβ, AR (mouse), calpeptin and ZVAD-fmk were purchased from Santa Cruz. Hsp90, phosphoAR and Hsp70 antibodies were purchased from BD Pharmingen. CHIP monoclonal antibody and MG132 were from Sigma Aldrich, USA. ECL detecting kit was from Thermo Scientific.
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