Skip to main content
. 2017 Oct 11;7(10):170115. doi: 10.1098/rsob.170115

Figure 3.

Figure 3.

PHF13 is degraded by the proteasome in a GSK3β-dependent manner early post-integration. (a) SupT1 cells were infected with equal amounts of VSVG pseudotyped HIV-1 NL4-3 IRES-eGFP. Simultaneously, cells were treated with 50 nM calpain Inhibitor I (ALLN), or at 6 hpi with 5 µM proteasome inhibitors lactacystin (LC) or MG132 for additional 6 h. PHF13, actin and p24 expression were analysed by immunoblotting. The same result was obtained in one additional experiment. (b) SupT1 cells were incubated with increasing amounts (0.1; 0.25; 0.5; 1 µM) of MLN4924, DMSO or were mock treated for 3 h. Subsequently cells were infected with 200 ng p24 VSVG pseudotyped HIV-1 NL4-3 IRES-eGFP. Forty-eight hours post-infection lysates were generated and analysed for PHF13, tubulin and p24 expression by immunoblotting. (c) SupT1 cells were treated with increasing amounts (10; 40; 100 µM) of GSK3β inhibitor SB216763 or 100 nM insulin for 6 h, followed by infection with 200 ng p24 VSVG pseudotyped HIV-1 NL4-3 IRES-eGFP. Forty-eight hours post-infection cells were analysed by immunoblot for PHF13, tubulin and p24 expression. (d) SupT1 cells were infected with 200 ng p24 VSVG pseudotyped HIV-1 NL4-3 IRES-eGFP, simultaneously cells were incubated with different drugs inhibiting various steps of HIV-1 replication. Forty-eight hours post-infection cells were lysed and subjected to immunoblot for the detection of PHF13, tubulin and HIV-1 p24. Raltegravir was used at 250 nM, whereas Efavirenz, Saquinavir and Flavopiridol were used at 50 nM. (e) SupT1 cells were infected with 200 ng p24 of VSVG pseudotyped HIV-1 NL4-3, the ΔVpr variant or HIV-1 with mutation D116N, blocking integration. Forty-eight hours later, cells were lysed and subjected to immunoblot for detection of PHF13, HIV-1 p24 and actin. The data presented in (b)–(e) were all confirmed in at least two additional independent replicates. Furthermore, for (a)–(d) the % of GFP+ cells was analysed in all experiments to control for equal infection rates. With Raltegravir and Efavirenz the number of infected (GFP+) cells was reduced to background levels.