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. 2020 Jun 4;219(7):e201908132. doi: 10.1083/jcb.201908132

Figure S2.

Figure S2.

LUZP1 and EPLIN interaction and functional studies. (A) FLAG-EPLINβ pulls down endogenous LUZP1. coIP experiments using protein extracts prepared from HEK293 cells stably expressing FLAG-BirA* (control) or FLAG-EPLINβ. The fusion proteins were immunoprecipitated using FLAG antibody-conjugated beads. FLAG, LUZP1, and actin antibodies were used to detect the FLAG fusions, endogenous LUZP1, and actin, respectively. (B) GFP-EPLINβ pulls down the LUZP1 truncation mutant lacking the N-terminal domains. coIP experiments using protein extracts prepared from HEK293T stable cells expressing FLAG-BirA* (control), FLAG-LUZP1, FLAG-LUZP1 (1-296aa) or FLAG-LUZP1 (297–1,076 aa), and GFP-EPLINβ. The GFP-fusion was immunoprecipitated using GFP antibody-conjugated beads. GFP and FLAG antibodies were used to detect the GFP and FLAG fusions, respectively. (C) FLAG-LUZP1 pulls-down actin. coIP experiments using protein extracts prepared from HEK293 cells stably expressing FLAG-BirA* (control) or FLAG-LUZP1. The fusion proteins were immunoprecipitated using FLAG antibody-conjugated beads. FLAG and actin antibodies were used to detect the FLAG fusions and endogenous actin, respectively. (D) LUZP1 depletion increases ciliation in RPE-1 cells. WT RPE-1 cells were transfected with control (NTsiRNA) or siRNAs targeting LUZP1and serum-starved for 72 h. Bar graph shows the mean percentage of ciliated cells (n > 200 cells per sample, three independent experiments) in RPE-1 cells transfected with the indicated siRNAs for 72 h. Error bars indicate SD. *, P < 0.05 (Student’s two-tailed t test). (E) Overexpressing LUZP1 or EPLIN causes a mild decrease in ciliation in RPE-1 cells. RPE-1 cells stably expressing GFP-only, GFP-LUZP1, GFP-EPLINα, or GFP-EPLINβ were serum starved for 72 h, after which the percentage of ciliated cells was determined. The graph shows data from three independent experiments. P values were determined by Student’s two-tailed t test. (F) LUZP1 and EPLIN silencing does not affect cell proliferation. RPE-1 cells were transfected with a control siRNA or siRNAs targeting LUZP1 or EPLIN. Cell confluence was assessed by time-lapse imaging (determined by phase-contrast images). Error bars represent SD.