A subset of Erg proteins labels endocytic hot spots. (A) Individual channels and merged epifluorescence micrographs of cells expressing the indicated Ergs tagged with mCherry (red) and/or GFP (green). (B) DIC, individual channels, and merged FMs of cells expressing Erg6-mCherry, stained with BODIPY (0.5 µg/ml). Arrowheads indicate cortical Erg6-mCherry dots with no detectable BODIPY signal. (C) Merged FMs of cells expressing Erg27-GFP and Abp1-mCherry, and merge of a time lapse of the indicated magnified areas showing repetitive assembly of endocytic actin (arrowheads) at ERSESs. (D and E) Immunoblots of anti-GFP agarose precipitates (IP [GFP]) from yeast expressing the indicated protein A (ProtA), GFP, or HA-tagged proteins (+) or nontagged versions (−), probed for HA (α-HA), Protein A (PAP), or GFP (α-GFP). 10 µg total protein was loaded in the inputs. (F) Ponceau red staining (upper panel) and immunoblot (lower panel) of glutathione-Sepharose pull-downs of GST or GST fused to the Osh2 ORD, expressed and purified from E. coli, and incubated with yeast extracts expressing Erg6-GFP (+) or the untagged Erg6 (−), probed for GFP (α-GFP). (G) Epifluorescence micrographs of the indicated yeast strains expressing GFP-D4H treated for 2 h with DMSO, TBF, FPM, or OSW-1. (H) Average ± SEM life span and Student’s t test P values of Sla1-GFP cortical patch life spans in osh2Δ cells expressing the indicated OSH2 alleles from centromeric plasmids. Significant differences are in red (n > 150). Kymographs of the time-lapse fluorescence movies for each experimental condition are shown. Images were taken every 0.5 s.