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)
Primer blast tool
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PRIMER-BLAST is a tool that assists users in designing and evaluating PCR primer sequences. It provides a platform to search for and analyze potential primer pairs targeting a specific gene or genomic region.
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2 protocols using primer blast tool
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Measuring PDGFRB Expression and Signaling
2
Microfluidic-based qPCR for RNA Expression
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A microfluidic-based qPCR dynamic array was used for the RNA expression analysis in samples derived from the Andalusian Public Health System Biobank cohort of patients.133 (link)–135 (link) Specific primers for human transcripts were designed with Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) and synthesized by Sigma-Aldrich (Table S2 ). Preamplification, exonuclease treatment and qPCR dynamic array based on microfluidic technology were implemented using the Biomark System (Fluidigm) by following the manufacturer’s instructions.136 (link) mRNA copy number of the transcripts analyzed were adjusted by normalization factor, calculated with the expression levels of ACTB, GAPDH and HPRT using geNorm 3.3 software.137 (link) Pearson correlation analyses were computed on mRNA expression levels and reported the values of the correlation coefficient (r) and two-tailed p values. The coefficient of determination (R2) was calculated from the Pearson correlation coefficient, and the best-fit line was plotted for each correlation.
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