Figure 4. Defining the mechanism of TDD... | Rockefeller University Press

Figure 4.

$Defining the mechanism of TDD inhibition.(A) Subcellular fractionation of ARPE cells expressing TDD into membrane and cytosolic fractions. (B) Subcellular fractions to enrich for TX-100–resistant TCVs from ARPE/HPV cells expressing TDD. (C) TIRFM imaging and quantification of clathrin dynamics in ARPE/HPV eGFP-CLCa cells indicates that TD phenocopies TDD by reducing the initiation density of bona fide CCPs to the same extent. Error bars are SD. (D) Cartoon showing the procedure used for immunoprecipitation of HA-TDD from ARPE cell lysate (with or without HA-TDD expression) using HA antibody and Protein G Sepharose 4 Fast Flow antibody purification resin. Mass spectrometry analysis was applied to the coIP samples for protein ID detection. (E) AP2 and SNX9 were identified as the only CME-related protein targets that were reproducibly enriched in the TDD coIPs. Values are average of three independent trials. ***, P ≤ 0.001.$

Defining the mechanism of TDD inhibition. (A) Subcellular fractionation of ARPE cells expressing TDD into membrane and cytosolic fractions. (B) Subcellular fractions to enrich for TX-100–resistant TCVs from ARPE/HPV cells expressing TDD. (C) TIRFM imaging and quantification of clathrin dynamics in ARPE/HPV eGFP-CLCa cells indicates that TD phenocopies TDD by reducing the initiation density of bona fide CCPs to the same extent. Error bars are SD. (D) Cartoon showing the procedure used for immunoprecipitation of HA-TDD from ARPE cell lysate (with or without HA-TDD expression) using HA antibody and Protein G Sepharose 4 Fast Flow antibody purification resin. Mass spectrometry analysis was applied to the coIP samples for protein ID detection. (E) AP2 and SNX9 were identified as the only CME-related protein targets that were reproducibly enriched in the TDD coIPs. Values are average of three independent trials. ***, P ≤ 0.001.