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. Author manuscript; available in PMC: 2023 Nov 21.
Published in final edited form as: Sci Signal. 2023 Oct 17;16(807):eadd6834. doi: 10.1126/scisignal.add6834

Fig, 3. Mutations in the Ptc PA binding motifs reduce Smo inhibition and Ptc binding to PA.

Fig, 3.

(A) S2 cells were transfected with Myc-SmoWT in combination with the indicated HA-tagged Ptc constructs. Identical volumes of lysate were immunoprecipitated (IP) and blotted (WB) with antibodies specific for Myc or HA. GFP served as transfection and lysate control. The density of each Myc-SmoWT band was quantified. N=3 independent experiments. *** indicates a p < 0.001 (Student’s t test). (B) PtcC interaction with lipids in a solid phase lipid binding assay. Membranes dotted with the indicated lipids were incubated with bacterially expressed and purified His-tagged PtcC or PtcCM124 and immunoblotted for His. C:M:H is a vehicle control. Blotting of a gel loaded with the same amount of His-PtcC and His-PtcCM124 applied to the lipid-dotted membranes (Input) is shown. N=3 independent experiments. (C) Bacterially expressed and purified His-PtcC or His-PtcCM124 was incubated with liposomes containing DOPA or DPPA and proteins that pelleted with liposomes were evaluated by Western blot. Control liposomes contained DOPC and POPE. Blots are representative of N=3 independent experiments. (D) Full-length HA-PtcWT or HA-PtcM124 was expressed in S2 cells, immunoprecipitated from lysates with the HA antibody, and incubated with liposomes with or without DPPA. Protein bound to the liposomes was detected by Western blot. The amount of Ptc in an equal amount of immunoprecipitation product is shown. N=3 independent experiments. The amounts of Ptc proteins bound to liposomes were quantified. ** indicates a p < 0.01 (Student’s t test). (E) Lysates of S2 cells transfected with HA-PtcWT with or without the HhN construct were immunoprecipitated with an antibody specific for HA and incubated with liposomes containing DPPA. Protein that bound to the liposomes was detected by Western blot. The amount of Ptc in an equal amount of immunoprecipitation product is shown (Input). N=3 independent experiments. Statistical analysis is shown in the Supplementary Materials (fig. S3E).