Figure 7

Mutant M1 binds less calnexin than wild-type E5 protein. HEK-293T cells were transfected with either (A-C) AU1-tagged codon-optimised HPV16 E5, the mutants M1, M2 and M3 or pcDNA 3.1 empty vector as control, (D and E) pEGFP-tagged HPV16 E5, mutant pEGFPM1, mock-control or pEGFP empty vector and lysed at 24 h posttransfection with CHAPS lysis buffer. A) Similar expression levels of all HPV16 E5 and the mutants M1, M2 and M3. B) Immunoprecipitations were performed using monoclonal anti-AU1, and proteins in the immune complex were detected using anti-AU1 and anti-calnexin. C) Quantification of co-precipitated calnexin for wild-type HPV16E5 protein, the mutants M1, M2, M3 and the vector control. The wild-type expression level was set to 100%. Data shown represent six independent experiments 2 plus standard errors of the mean. P values were calculated with paired two-tailed Student's t-test. D) Similar expression levels of pEGFP-HPV16-E5 and the mutant pEGFP-M1. E) Immunoprecipitations were performed using monoclonal anti-GFP, and proteins in the immune complex were detected using anti-GFP and anti-calnexin. Molecular-mass markers in kDa are indicated at the left of the blots.