Fig. 3

Overexpression of mortalin promotes HIV-1 Tat degradation by ubiquitination. a) Percentage change in mRNA expression of mortalin in indicated trasnfected group analyzed by RT-PCR, GAPDH was used as internal control (n = 5). b) Percentage change in the expression of mortalin at protein level in indicated transfected groups, c) analyzed by western blot, GAPDH was taken as loading control (n = 5). d) Representative immunofluorescence image showed the expression of exogenous mortalin tagged with V5 and HIV-1 Tat in all transfected PDA after 24 h of transfection, scale bar 20 μm (n = 3). e) Representative western blot showed the expression of HIV-1 Tat protein in transfected cell upon treatment with MG132, indicated by “+” and “-” represents presence and absence of MG132 (n=3). GAPDH was used as loading control, and lower panel of western blot represents the expression of HIV-1 Tat in trasnfected cell without MG132 (n = 4). f) Pictorial representation of docked molecule of HIV-1 Tat and mortalin of substrate-binding site. g, h) Immunoblots of coimmuniprecipitated PDA of input (control). PC (plasmid control), HIV-1 Tat, and cotransfected (mortalin and HIV-1 Tat) protein probed with anti-mortalin and anti-Tat antibodies, h) Represent the reverse CO-IP (n = 4). All data represent mean ± standard deviation, from independent experiments (n stands for number of independent experiments), *p< 0.05, **p< 0.005 with respect to control, #p< 0.05, #p< 0.005 with respect to HIV-1 Tat and contransfected group (PC stands for pasmid control and PC+PC is used as plasmid control in cotransfected cells)