FAM102A and FAM102B bind PI(4,5)P 2 at the PM through their C2 domain. (A and B) C2FAM102A-EGFP disassociates from the PM after the recruitment of a 5-phosphatase (INPP5E) but not after the recruitment of a 4-phosphatase (Sac1). The PI(4,5)P2 probe iRFP-PHPLCδ1 and the PI4P probe iRFP-P4M were used as positive controls to indicate the successful depletion of each phosphoinositide. The peak fluorescence intensity of C2 FAM102A -EGFP on the PM was measured both before and after phosphatase recruitment and quantified as a ratio, as shown in B. Unpaired t test. n = 14 cells for Sac1 and n = 13 cells for INPP5E from three independent experiments. Mean ± SEM. (C and D) C2 FAM102B-EGFP was tested as in A and quantified as in D. Unpaired t test. n = 17 cells for Sac1 and n = 16 cells for INPP5E from three independent experiments. Mean ± SEM. (E and F) U2OS cells stably expressing BLTP2^Halo were transfected with either FAM102A-EGFP (E) or FAM102B-EGFP (F). The focal plane was adjusted to optimize visualization of ER-PM contacts at the basal surface. In both FAM102A and FAM102B co-expressing cells, BLTP2^Halo disassociated from the PM after PI(4,5)P2 but not after PI4P depletion. (G and H) In COS-7 cells co-expressing BLTP2^Halo, the muscarinic M1R receptor, and either FAM102A-GFP (G) or FAM102B-GFP (H), BLTP2^Halo disassociates from ER-PM contact sites and redistributes to the entire ER in response to PI(4,5)P2 depletion by Oxo-M addition. BLTP2^Halo relocalizes to ER-PM contacts after the addition of the Oxo-M antagonist atropine.