Ras (D2C1) Rabbit mAb #8955

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	-Rabbit (1:1000)

Protocol tips
-Rabbit (1:1000)

Publication protocol

Cells were lysed in RIPA buffer (Boston Bioproducts) containing Halt protease and phosphatase inhibitor cocktail (Thermo Fisher). For experiments involving dox-inducible constructs, lysates were cleared by centrifugation, and protein concentration determined by Pierce BCA protein assay kit (Thermo Fisher). Samples were denatured by boiling in loading buffer (Cell Signaling). 20 μg of lysates were loaded on 10% MiniProtean TGX gels (Bio-Rad), transferred to Immun-Blot PVDF membranes (Bio-Rad), blocked in TBST (0.1% Tween-20) and 5% milk. For all other experiments, samples were denatured by boiling in loading buffer (BioRad) and 25 μg of lysates were loaded on 4–12% Bis-Tris gradient gels (Thermo Fisher), run using MOPS buffer, transferred to Immobilon-P PVDF membranes (Millipore) and blocked in TBST (0.1% Tween-20)/5% BSA (Sigma).
Primary incubation with antibodies was performed overnight at 4° in 5% BSA, followed by appropriate HRP-conjugated secondary antisera (Santa Cruz Biotechnology) and detected using ECL (Thermo Fisher). Antibodies were obtained from Cell Signaling and raised against the following proteins: phospho p-38 (4511), p38 (8690), p-p44/p42 (ERK1/2) (9101), p44/p42 (ERK1/2) (4695), p-SAPK/JNK (4668), SAPK/JNK (9252), P-EGFR (3777, 2234), EGFR (2232), KRAS (8955), PARP (9542), cleaved-PARP (5625), α-Tubulin (3873) and β-Actin (3700, 4970). Additionally, we used an antibody against GFP (A-21311, Thermo Fisher), DUSP1 (ab1351, abcam) and DUSP6 (ab76310, abcam and SC-377070, SC-137426, Santa Cruz)..

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Manufacturer protocol

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