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. 2012 Nov 29;8(11):e1003048. doi: 10.1371/journal.ppat.1003048

Figure 4. Hsp90 inhibitors induce proteasomal degradation of LANA.

Figure 4

(A) Half-life analysis of LANA termini. N- or C- termini of Flag-tagged LANA vectors were transfected separately into HeLa cells for 16 hours, followed by cycloheximide (CHX) treatment for 0, 3, 6, 12, 24 and 48 hours. Whole cells lysates were immunoblotted with anti-Flag antibody. β-Actin was used for loading control. (B) LANA degradation induced by 17-DMAG. HeLa cells were transfected with full-length LANA overnight, followed by incubation with vehicle or 0.5 µM 17-DMAG in the presence of 50 µg/ml cycloheximide for 0, 3, 6, 12, 24 and 48 hours respectively, whole cells lysates were immunoblotted with anti-LANA. (C–D) Quantitative analysis of the above results. (E–F) LANA degradation inhibited by MG-132 or Lactacystin. After transfection with LANA vector, Hela cells were treated with no drug or 17-DMAG (1 µM) for 24-hours in the absence (−) or presence (+) of proteasome inhibitor MG-132 (10 µM) for the last 6 hours or Lactacystin (10 µM) for 24 hours, whole cells lyses were immunoblotted with anti-LANA antibody. (G) Proteasomal degradation of LANA in BCBL-1 cells. BCBL-1 were treated for 24 hours with no drug or AUY922 (0.1 µM) in the absence (−) or presence (+) of proteasome inhibitor MG-132 (10 µM) for the last 6 hours, whole cells lysates were immunoblotted with anti-LANA antibody. (H) Poly-ubiquitinated degradation of LANA. HeLa cells were transfected with LANA, followed by treatment with no drug or 1 µM 17-DMAG for 24 hours in the presence of DMSO or 10 µM MG-132 for the last 6 hours. Cells lysates were immunoprecitated with rat anti-LANA antibody, followed by immunoblotting with rabbit anti-ubiquitin and anti-LANA antibodies, the bracket shows the poly-ubiquitinated LANA (Ub-LANA). (I) Immunofluorescence analysis of LANA degradation. L1T2 cells were treated with no drug or 1 µM 17-DMAG for 48 hours, incubated with primary anti-LANA (rabbit) and after fixation and permeabilization, stained with anti-rabbit Texas-Red conjugated antibodies (red, LANA), nuclei were stained with DAPI.