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Fig. 1 | Experimental Hematology & Oncology

Fig. 1

From: Proteomic analysis of HEK293 cells expressing non small cell lung carcinoma associated epidermal growth factor receptor variants reveals induction of heat shock response

Fig. 1

Induction of heat shock response to EGFR mutants and regulation of Hsp70 by L861Q mutant. HEK293 stable cell lines expressing each mutant and wild type receptor were seeded in 35 mm dishes in duplicates. At 80-90 % confluency, cells were serum starved overnight followed by EGF stimulation at 10 ng/ml. At 10 min post stimulation, cells were harvested. Total protein from one set of plates and total RNA from duplicate set of plates were recovered. Equal amount of protein recovered from each receptor expressing cells was subjected to western blot analysis using anti phospho EGFR antibody specific to autophosphorylation site, tyr1068 residue. Blot was striped and re-probed with total EGFR antibody. RNA samples were reverse transcribed and subjected to quantitative real time PCR using gene specific primers. For Hsp70 expression studies, L861Q mutant expressing cells were cultured, serum starved followed by EGF stimulation. At every 5 min interval, total RNA and protein lysates recovered were subjected to quantitative real time PCR using Hsp70 primers and western blot analysis with anti Hsp70 antibody respectively. For inhibitory studies; cells were serum starved, treated with different concentrations of Gefitinib followed by EGF stimulation. Western blot analysis was done using anti Hsp70, anti phospho and total EGFR antibodies. a Phosphorylation status of three mutants vs. wild type receptor. b Folds difference of mutants vs. wild type receptor regulated gene transcripts of few heat shock proteins. c Hsp70 expression at transcript level measured at every 5 min following EGF stimulation in L861Q mutant vs. wild type receptor expressing cells d Hsp70 expression at protein level for 20 min, Hsp70 and phospho EGFR levels in cells treated with TKI, Gefitinib

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