Fig 1: CD2AP/CIN85 promotes actin-branched network formation around the base of primary cilia.A and B, RPE1 cells transfected with EHBP1L1 #1 siRNA or cotransfected with CD2AP and CIN85 siRNAs were stained with antibodies against Arp2 (A) and PCNT (A and B) or with Alexa-568 phalloidin (B). Dotted circles indicate a 1 µm radius around the PCNT-labeled basal body. Magnified images of the circled areas are shown in the insets. Scale bar represent (main images) 2 µm; (insets) 0.5 µm. D and E, fluorescence intensities of Arp2 and Alexa-568 phalloidin within a 1 µm radius around the basal body were measured and plotted against siRNA-treated cells (control, CD2AP/CIN85, or EHBP1L1 #1 and #2). n = 50 for each experiment. Error bars represent S.D. p values were calculated using Student’s t test. Statistical significance was set at ***p < 0.001. C, cell lysates treated with siRNAs (control, CD2AP/CIN85, or EHBP1L1 #1 and #2) were immunoblotted with EHBP1L1, CD2AP, CIN85, and Arp2 antibodies. GAPDH was used as the loading control. PCNT, pericentrin; RPE, retinal pigment epithelium.
Fig 2: EHBP1L1 negatively regulates ciliary length.A, RPE1 cells were transfected with control (Ctrl) or two EHBP1L1 siRNAs (#1 and #2). EHBP1L1 protein depletion was analyzed by immunoblotting using an EHBP1L1 antibody. GAPDH was used as the loading control. B, cells treated with control or EHBP1L1 siRNAs (#1 and #2) were immunostained with ac-tubulin (green) and pericentrin (PCNT) (red) to visualize primary cilia and basal bodies, respectively. Scale bar represents 5 µm. C, ciliary lengths, as indicated by ac-tubulin in control or EHBP1L1 siRNA-transfected cells (#1 and #2), were measured and are indicated on the graph (n = 241–277 cells). Error bars represent the S.D. p values were calculated using ordinary one-way ANOVA followed by Tukey’s test. Statistical significance was set at ***p < 0.001. D, schematic drawing of full-length FLAG-tagged EHBP1L1 and deletion mutants used for the rescue experiment. Orange triangles indicate the proline-X-X-proline (PXXP) motif. E, EHBP1L1-depleted cells (siEHBP1L1) were expressed with or without (-) FLAG-tagged full-length EHBP1L1 and deletion mutants (Full, ?PR, ?CH, and ?C2). Flag-positive ciliary length was measured and is indicated on the graph. The length of endogenous EHBP1L1-positive cilia in control cells (EHBP1L1+) is shown for comparison. Ciliary length was measured using Arl13b signal (n = 72–159 cells). Error bars represent the S.D. p values were calculated using ordinary one-way ANOVA followed by Tukey’s test. Statistical significance was set at ***p < 0.001. bMERB, bivalent Mical/EHBP Rab binding domain; C2, Ca2+/phospholipid-binding domain; CH, calponin homology domain; PR, proline-rich region; RPE, retinal pigment epithelium.
Supplier Page from MilliporeSigma for Anti-PCNT antibody produced in rabbit