Fig. 5

TM4SF5-dependent downregulation of SLAMF7 via lysosomal trafficking. a SNU761 cells expressing FLAG-SLAMF7 and HA-EV or HA-TM4SF5 were incubated at 4 °C and then at 37 °C for the indicated times. Cells were harvested and processed for immunoblots using the indicated antibodies. b, c SNU761 cells were transfected with HA-TM4SF5 and FLAG-SLAMF7 and immunostained with HA, FLAG, and LAMP1 or ZO-1 antibodies. Colocalization coefficients were determined by Pearson’s correlation (b). Each dot depicts a correlation value per cell in one of three independent experiments. Representative images are shown (c). d SNU761 cells were transfected with FLAG-SLAMF7 and HA-EV or HA-TM4SF5 and treated with ST-5-002 with or without chloroquine (CQ, 10 μM) for 24 h. Cells were harvested for immunoblots with the indicated antibodies. Scale bar, 100 μm. e–h SNU761 cells were transfected with Strep-TM4SF5 and FLAG-SLAMF7 and treated with ST-5-002 (e) or ST-3-001 (f) for 24 h. Cells were harvested and lysed with Brij58-containing lysis buffer. Whole-cell lysates were subjected to streptavidin pulldown and immunoblots. SNU761 cells were transfected with HA-TM4SF5 and FLAG-SLAMF7 and treated with ST-5-001 or ST-5-002 (2.5 μM, 24 h). Cells were fixed with ice-cold methanol and immunostained with the indicated antibodies. Representative images are shown (g). Colocalization coefficients were determined by Pearson’s correlation. Scale bars, 100 μm (h). Each dot depicts a correlation value per cell in one of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001, ns non-significant, unpaired Student’s t test or Two-way ANOVA. Data is represented as the mean ± SEM. Data represent three isolated experiments. See also Fig. S6