Anti pan ras

Detection of protein expression by immunoblotting was performed using anti-CDC42 (Cell Signaling Technology), anti-β-tubulin, anti-pan-Ras, and anti-p16 (Santa Cruz Biotechnology).
ON-TARGETplus SMARTpool siRNA targeting RHOA, RAC1, RAC2, CDC42, and the nontargeting (control) pool were transfected into senescent IMR-90 cells with Dharmafect 1 reagent (all from Dharmacon). Cells were washed 24 h after transfection; transfer assay, cell viability assay, and evaluation of knockdown were performed 48 h later.
For retroviral transductions, the following plasmids were used: pLPC mCherry or pLPC EGFP (mCherry or GFP fused to the gene of interest from an internal CMV promoter with a puromycin resistance gene driven by the LTR). Cells were infected with mCherry-H-Ras^12V, GFP, or mCherry alone. Retrovirus-mediated gene transfer was performed as previously described.
Total RNA was isolated using a NucleoSpin kit (Macherey Nagel) and reverse-transcribed using the RevertAid H Minus first strand cDNA synthesis kit (Fermentas). The cDNA samples were used for real-time PCR (Fast SYBR Green master mix, Applied Biosystems). PCR amplifications were carried out using StepOnePlus real-time PCR systems (Applied Biosystems). The relative expression of each gene was normalized using the expression levels of GAPDH.

Mouse monoclonal anti-hemagglutinin (HA) and rabbit polyclonals anti-RhoGDI, anti-HRAS, anti-panRAS, anti-PEA15, anti-RSK1 anti–p-ERK, and anti-ERK2 were from Santa Cruz Biotechnology, Santa Cruz, CA. Rabbit polyclonal anti-p-RSK-1, anti-ELK1, and anti-p-ELK1 were from Cell Signaling, Billerica, MA. Rabbit polyclonal anti-caveolin and anti–lamin A were from BD Biosciences, San Jose, CA. Mouse monoclonal anti–transferrin receptor was from Zymed Laboratories, Waltham, MA. Rabbit polyclonal anti-calreticulin was from Calbiochem.