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. 2021 Jan 7;220(2):e201902073. doi: 10.1083/jcb.201902073

Figure 5.

Figure 5.

pY374-PKCδ and FER inhibit release of RAB5 from the nascent late endosome. (a–d) Time course of RAB11 and RAB7 recruitment to RAB5-positive endosomes. Representative confocal images of the indicated RAB immunofluorescence 360 min after initiating trafficking by EGF (20 ng/ml) stimulation in pCas9Vec or PTPN14-KO cells (a and c; arrowheads indicate colocalization) and quantification from confocal images of the colocalization of RAB5 (Alexa Fluor 488) immunofluorescence with either RAB11 (b; Alexa Fluor 594) or RAB 7 (d; Alexa Fluor 594) immunofluorescence at the indicated times after EGF stimulation (mean ± SEM; n > 30 cells; **, P < 0.01 using Student’s t test). (e) PLAs of RAB5 and RAB7 after 360-min EGF (20 ng/ml) stimulation in pCas9Vec or PTPN14-KO (two different clones, a and o) cells (mean ± SEM; n = 3; *, P < 0.05; **, P < 0.01 using Student’s t test). (f) Colocalization of RAB5 and RAB7 immunofluorescence 360 min after EGF (20 ng/ml) stimulation to induce trafficking in pCas9Vec or PTPN14-KO cells transiently transfected with control (siCtrl) or PKCδ (siPKCδ) siRNAs, quantified from confocal images (not shown; mean ± SEM; n > 30 cells; *, P < 0.05, using Student’s t test). (g) PLA of RAB5 and RAB7 after 360-min EGF (20 ng/ml) stimulation in pCas9Vec or PTPN14-KO transiently transfected with siCtrl, siFER3, or siFER4 siRNAs after 360 min of EGF stimulation to induce trafficking (mean ± SEM; n = 3; *, P < 0.05 using Student’s t test).