Fig. 4 | Signal Transduction and Targeted Therapy

Fig. 4

From: Isoxazole-based molecules restore NK cell immune surveillance in hepatocarcinogenesis by targeting TM4SF5 and SLAMF7 linkage

Fig. 4

Binding of TM4SF5 to SLAMF7 depending on N-glycosylation causes lysosomal degradation of SLAMF7. a, b Huh7KO-HA-EV or Huh7KO-HA-TM4SF5 cells were treated with conditioned media (CM) of NK92-EV or NK92-TM4SF5 cells (a) or vice versa (b) for 24 h before whole-cell lysates were prepared and immunoblotted for the indicated molecules. c, d Subconfluent SNU761 cells expressing Strep empty vector (Strep-EV) or Strep-TM4SF5 (TM) were treated without (c) or with (d) DMSO or ST-5-001 were harvested. Cell lysates were pulled down using streptavidin-agarose beads and immunoblotted for SLAMF7. Different amounts of input proteins were loaded for SLAMF7 immunoblotting to see clear coimmunoprecipitation of endogenous SLAMF7 (c). e, f SNU761 cells were transiently transfected with WT or various SLAMF7 mutants of N-glycosylation residues (N98, N142, N148, N172, N176, and/or N204), prior to immunoblots for the indicated molecules (e) or for coimmunoprecipitation (f). g SNU761 cells transfected with FLAG-SLAMF7 and Strep-TM4SF5 WT or Gly– were harvested and lysed with Brij58-containing lysis buffer. Whole-cell lysates were subjected to streptavidin pulldown and immunoblot. h, i SNU761 cells transfected with Strep-TM4SF5 and FLAG-SLAMF7 deletion mutants (h) or N-glycosylation mutants (i) were harvested and lysed with Brij58-containing lysis buffer. Whole-cell lysates were subjected to streptavidin pulldown and immunoblot. ΔECD: Δ1–225; ΔTM: Δ226–247; NQ#6:N204Q; NQ#45:N172/176Q; NQ#456:N172/176/204Q. * in the immunoblots indicate non-specific bands. j SNU761 cells were transfected with HA-TM4SF5 together with FLAG-SLAMF7 WT, NQ#6, or NQ#456. SLAMF7 with N-glycosylation mutations; NQ#1; N98Q, NQ#2; N142Q, NQ#3; N148Q, NQ#123; N98/142/148Q, NQ#456; N1172/176/204Q, NQ#1456; N98/172/176/204Q, NQ#2456; N142/172/176/204Q, NQ#23456; N142/148/172/176/204Q. Cells were randomly stained with HA (blue), FLAG (green), and LAMP1 (red). Images were randomly captured by a confocal microscope. Data represent three independent experiments. See also Fig. S5

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