Fig. 3: ORP4L/OSBP transfer PI(4)P from Golgi to PM for PI(4,5)P2 biosynthesis.
a Kinetic analysis of PI(4)P binding to ORP4L (upper) and OSBP (lower) determined by SPR in real-time. Mean KD values ± SD (n = 3 biological replicates) are indicated. b, c Time course of DsRed-OSBP protein, the PI(4)P probe GFP-P4MSidM and the cholesterol probe mCherry-D4H at the PM and Golgi of ORP4L KI T-cells. Quantification of fluorescent signals in the PM (c, left) and Golgi (c, right) are shown. Scale bar, 5 μm. d Relative fluorescence intensity changes of the GFP-P4MSidM, mCherry-D4H and GFP-PHPLCδ1 in the PM of ORP4L KI T-cells subjected to OSBP knockdown and re-expression with wild-type OSBP or OSBP[△PI(4)P] or OSBP(△ORP4L). e Relative fluorescence intensity changes of GFP-P4MSidM, mCherry-D4H and GFP-PHPLCδ1 in the PM of ORP4L KI T-cells subjected to ORP4L knockdown and re-expression of wild-type ORP4L or ORP4L(△OSBP). f Relative fluorescence intensity changes of GFP-P4MSidM in the PM of ORP4L KI T-cells upon VAPA knockdown. g, h Relative fluorescence intensity changes of GFP-P4MSidM in the PM of ORP4L KI T-cells subjected to OSBP knockdown and re-expression with wild-type OSBP or OSBP(△VAPA) (g), or subjected to ORP4L knockdown and re-expression with wild-type ORP4L or ORP4L(△VAPA) (h). i, j Relative fluorescence intensity changes of GFP-P4MSidM (i) and mCherry-D4H (j) in PM of ORP4L KI T-cells pre-treated with or without 5 nM OSW-1 for 1 h. k, l Relative fluorescence intensity changes of GFP-P4MSidM (k) and GFP-PHPLCδ1 (l) in PM of ORP4L KI T-cells pre-treated with or without 0.5 mM serine and 200 µM D609 for 12 h. m, n Relative fluorescence intensity changes of the GFP-P4MSidM (m) and GFP-PHPLCδ1 (n) in PM of ORP4L KI T-cells subjected to ORP4L knockdown and then treated with or without 0.5 mM serine and 200 µM D609 for 12 h. In panel (c–n), data are presented as Mean ± SD (n = 10 cells from one mouse). The same experiments were repeated twice in T-cells from two mice. Source data are provided as a Source Data file.
