Fig. 1: AP-1 loading of STING into CCVs at the TGN.
From: Clathrin-associated AP-1 controls termination of STING signalling
a, Whole-cell lysate (WCL) and CCV fractions from HeLa cells expressing Flag-tagged STING (HeLaSTING) were analysed by western blot. Clathrin heavy chain (CHC) and GAPDH were used as loading and processing controls. b, Airyscan imaging of HeLaSTING cells stimulated with diABZI. One representative cell of n = 7 cells. White arrows point at occurrences of pSTING. Scale bars, 4 µm (left) or 1 µm (magnified panels). c, Quantification of the colocalization of pSTING with CHC described in b, using Manders’ colocalization coefficients. Mean ± s.e.m. of n = 7 cells. d, STED images showing pSTING enclosed in CCVs from cells transfected with mCherry–clathrin and Flag–STING and stimulated with diABZI. Regions 1 and 2 are magnified from a large-field-of-view STED image (see Extended Data Fig. 2a). Scale bars, 200 nm. e, CLEM of HeLaGFP–STING cells stimulated with diABZI. The images depict box 3 of Extended Data Fig. 2b in Airyscan microscopy (left) or electron microscopy at different Z-heights (three right panels). White arrows indicate CCVs. Scale bars, 0.5 µm. f, WCL and CCV fractions from HeLa cells that were treated with non-targeting control (NC) small interfering RNA (siRNA) or AP-1 siRNA and stimulated with diABZI were analysed by western blot. CHC was used as a loading control. g, Quantification of the area of pSTING in bright-field fluorescent microscopy images of HeLaSTING cells that were treated with siRNAs and stimulated with diABZI. Mean ± s.e.m. of n = 3 independent experiments with 99 fields of view per condition. h, HeLa cells transfected with siRNAs and stimulated with diABZI were analysed by western blot. Ratios of STING versus loading control (GAPDH) normalized to the 0-h time point of each condition. One representative example of three (a–c,h) or two (f) independent experiments is shown.
