FIGURE 6.

TMEM184A knockdown blocks heparin responses. A, RAOECs (not transfected) were evaluated for TMEM184A staining (NTD antibody) over a range (a–d) of cell densities. Scale bars = 10 μm. RAOECs were transfected with control or TMEM184A shRNA constructs and seeded on coverslips as described under “Experimental Procedures.” TMEM knockdown was confirmed by staining some cells from each experiment for TMEM184A using the NTD antibody (e.g. B, images from permeabilized cells). Results from three experiments with more than 200 cells for each condition are shown in the graph. After 72 h, cells were treated with TNFα (25 ng/ml) with or without a 20 min pretreatment of 200 μg/ml heparin, fixed, and stained for pp38 (C) or pJNK (D). Nuclear levels of the activated enzymes were determined. E, TMEM184A or control shRNA-transfected cells were treated with 200 μg/ml heparin for up to 30 min, fixed, and stained for DUSP1, and then nuclear levels of DUSP1 were determined. F, TMEM184A or control shRNA-transfected cells were treated for 60 min with TNFα (25 ng/ml) with or without a 20 min pretreatment of 200 μg/ml heparin and stained with Alexa Fluor 488-phalloidin. Fluorescence peaks were determined as in Fig. 5. All graphical results are from at least three repeat experiments per condition with at least 50 cells analyzed for each experiment. †, p < 0.05.