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. 2002 Apr;13(4):1252–1262. doi: 10.1091/mbc.01-05-0235

Figure 5.

Figure 5

Figure 5

The majority of PLD1b is Triton insoluble. (A) HA-tagged PLD1b was immunoprecipitated from antigen-stimulated RBL-2H3 cells cotransfected with HA-tagged PLD1b and GFP-tagged ARF6. After electrophoresis the gel was probed with an anti-ARF6 antibody. WCL, whole cell lysate; IP, immunoprecipitated sample (The IP lane is equivalent to ∼9 times more loading than the WCL lane). (B) RBL-2H3 cells were cotransfected with HA-tagged PLD1b and GFP-tagged versions of both Rac1 and PKCα. After separation on a 4–20% gradient gel, Rac1 was detected with an anti-Rac1 antibody (Upstate Biotechnology) PKCα with an anti-PKCα antibody (Affinity BioReagents, Golden, CO) and PLD1b with the anti-HA antibody. P, Pellet/Triton-insoluble fraction; S, Supernatant/Triton-soluble pellet (The P lane is equivalent to ∼9 times more loading than the S lane). Only the bands corresponding to endogenous proteins are shown in the cases of Rac1 and PKCα (overexpressed proteins showed a similar pattern). (C) RBL-2H3 cells were transfected with HA-tagged PLD1b as indicated by the red staining. F-actin was detected with FITC-conjugated phalloidin as indicated by the green staining. Yellow staining indicates regions of colocalization between PLD1b and F-actin. Numbering down the left-hand side indicates time after addition of 50 ng/ml DNP-HSA. Similar results were obtained in at least three experiments.