Fig 2. Extracellular rhHSP70 modules membrane level and phosphorylation of Cx43.

A. Immunofluorescence detection of Cx43 (green) in HMEC after treatment with 5μg/ml rhHSP70 for indicated times (DAPI staining of nuclei). Arrows indicate Cx43 plaques. Representative of 5 experiments. Bar 20 μm. B. Western blot of the total and membrane fraction (Triton X-100 insoluble) of Cx43. P0, P1 and P2 denotes the three major Cx43 migration bands. Cell membrane lysates immunoblotted for Cx43, after treatment with rhHSP70 for time periods as indicated (Hsc70 as loading control). Right panel shows changes in band intensity of the membrane fraction related to the total Cx43 expression level (mean ± SD, n=5; **P<0.01, *P<0.05 vs control [t=0 min] in all cases). C. Effect of rhHSP70 on Cx43 phosphorylation pattern. Western blots using three different antibodies against the carboxy terminal part of Cx43 to detect phosphorylation on serine at position Ser262, Ser255 and Ser368 (representative of 5 experiments). D. rhHSP70 leads to phosphorylate Cx43 in a TLR4-dependent manner. Western blot showing phosphorylation on Ser368. When indicated, cells were pre-treated for 60 min with polymyxin B (PMB10 μM) or the neutralysing anti-TLR4 (AbTLR4 10 μg/ml). rhHSP70 was (or not) added for 60 min (representative of 5 experiments). Histogram shows changes in the phosphorylated status of Cx43 in response to 60 min of cell treatment with rhHSP70 (black) or rhHSP70 plus AbTLR4 (grey), expressed as percentage of control (mean ± SD, n=5; **P<0.01, *P<0.05 vs control). E. Immunofluorescence detection of ZO-1 in HMEC after exposure to rhHSP70 for indicated times. Representative of 5 experiments. Cell nuclei stained with DAPI. Bar 20 μm. F. Coimmunoprecipitation of Cx43 and ZO-1 in HMEC, stimulated or not by rhHSP70 for time periods as indicated. The total Cx43 shows slight variations in the unphosphorylated form P0 and the phosphorylated forms P1 and P2 (Hsc70 as loading control; representative of 4 experiments).