rabbit anti ha  (Cell Signaling Technology Inc)

Journal: The Journal of Cell Biology

Article Title: The intercentriolar fibers function as docking sites of centriolar satellites for cilia assembly

doi: 10.1083/jcb.202105065

Figure Lengend Snippet: Generation of the CROCC KO RPE1 cells. (A) The CROCC KO RPE1 cells were coimmunostained with antibodies specific to γ-tubulin (magenta) and CROCC (cyan). (B) The CROCC KO cells were subjected to immunoblot analyses with antibodies specific to CROCC, PCM1, CEP250, CEP68, and GAPDH. (C) The number of cells with cilia was counted. (D) The distance between the centrioles in CROCC KO cells was determined after the treatment of 20 μM nocodazole for 2 h. (E) The CROCC KO cells were coimmunostained with antibodies specific to CEP68 (cyan) and centrin-2 (magenta). (A and E) Scale bars, 10 μm; inset scale bars, 2 μm. (C and D) More than 30 cells per group were counted in three independent experiments. Graph values are expressed as mean and SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test (*, P < 0.05; **, P < 0.01; n.s., not significant). Source data are available for this figure: .

Article Snippet: Rabbit anti-CROCC (HPA021191; IS, 1:200; IB, 1:300; Sigma-Aldrich), mouse anti-acetylated tubulin (T6793; IS, 1:200; Sigma-Aldrich), mouse anti-α-tubulin (T6199; IS, 1:1,000; IB, 1:10,000; Sigma-Aldrich), goat anti-FLAG (ab1257; IS, 1:500; IB, 1:2,000; Abcam), rabbit anti-CEP290 (ab84870; IS, 1:100; IB, 1:200; Abcam), rabbit anti-OFD1 (ab97861; IS, 1:100; IB, 1:100; Abcam), mouse anti-FLAG (F3165; IS, 1:2,000; IB, 1:20,000; Sigma-Aldrich), rabbit anti-γ-tubulin (ab11317; IS, 1:300; Abcam), rabbit anti-centrin-2 (04-1624; IS, 1:500; Millipore), mouse anti-γ-tubulin (ab11316; IS, 1:300; Abcam), rabbit anti-CEP68 (15147-1-AP; IS, 1:100; IB, 1:500; Proteintech), rabbit anti-CEP72 (A301-297A; IS, 1:500; IB, 1:500; Bethyl), and mouse anti-GAPDH (AM4300; IB, 1:10,000; Invitrogen) antibodies were purchased from commercial suppliers.

Techniques: Western Blot

Journal: The Journal of Cell Biology

Article Title: The intercentriolar fibers function as docking sites of centriolar satellites for cilia assembly

doi: 10.1083/jcb.202105065

Figure Lengend Snippet: Generation of the CEP250 KO RPE1 cells. (A) The CEP250 KO RPE1 cells were coimmunostained with antibodies specific to centrin-2 (magenta) and CEP250 (cyan). (B) The CEP250 KO cells were subjected to immunoblot analyses with antibodies specific to CEP250, PCM1, CROCC, CEP68, and GAPDH. (C) The number of cells with cilia was counted. (D) The number of cells with centriole disjunction (>2 μm) was counted. (E) The CEP250 KO cells with and without cilia were coimmunostained with antibodies specific to CROCC (cyan) and acetylated tubulin (magenta). (F) The number of cells with centrosome/basal body CROCC signals was counted in CEP250 KO cells with and without cilia. (G) The CEP250 KO cells were cultured in serum-deprived medium for 48 h to induce cilia assembly and subjected to coimmunostaining analysis with antibodies specific to CROCC (cyan) and acetylated tubulin (magenta). (H) The number of cells with CROCC fibers was counted in cells. (A, E, and G) Scale bars, 10 μm; inset scale bars, 2 μm. (C, D, F, and H) More than 30 cells per group were counted in three independent experiments. Graph values are expressed as mean and SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test (**, P < 0.01; ***, P < 0.001; n.s., not significant). Source data are available for this figure: .

Article Snippet: Rabbit anti-CROCC (HPA021191; IS, 1:200; IB, 1:300; Sigma-Aldrich), mouse anti-acetylated tubulin (T6793; IS, 1:200; Sigma-Aldrich), mouse anti-α-tubulin (T6199; IS, 1:1,000; IB, 1:10,000; Sigma-Aldrich), goat anti-FLAG (ab1257; IS, 1:500; IB, 1:2,000; Abcam), rabbit anti-CEP290 (ab84870; IS, 1:100; IB, 1:200; Abcam), rabbit anti-OFD1 (ab97861; IS, 1:100; IB, 1:100; Abcam), mouse anti-FLAG (F3165; IS, 1:2,000; IB, 1:20,000; Sigma-Aldrich), rabbit anti-γ-tubulin (ab11317; IS, 1:300; Abcam), rabbit anti-centrin-2 (04-1624; IS, 1:500; Millipore), mouse anti-γ-tubulin (ab11316; IS, 1:300; Abcam), rabbit anti-CEP68 (15147-1-AP; IS, 1:100; IB, 1:500; Proteintech), rabbit anti-CEP72 (A301-297A; IS, 1:500; IB, 1:500; Bethyl), and mouse anti-GAPDH (AM4300; IB, 1:10,000; Invitrogen) antibodies were purchased from commercial suppliers.

Techniques: Western Blot, Cell Culture

Journal: The Journal of Cell Biology

Article Title: The intercentriolar fibers function as docking sites of centriolar satellites for cilia assembly

doi: 10.1083/jcb.202105065

Figure Lengend Snippet: Expression of FLAG-CROCC FL and FLAG-CROCC 303–1741 in CROCC KO RPE1 cells. (A) Ectopic expression of FLAG-CROCC FL and FLAG-CROCC 303-1741 were induced with 1 μg/ml doxycycline for up to 4 h in the CROCC KO cells. The cells were coimmunostained with antibodies specific to FLAG (cyan) and Centrin-2 (magenta). Scale bar, 10 μm. (B) The cells were immunoblotted with antibodies specific to FLAG, CROCC, and GAPDH. (C) Intensities of PCM1 at the centrosomes were determined. More than 30 cells per group were counted in three independent experiments. Within each box, the black center line represents the median value, the black box contains the interquartile range, and the black whiskers extend to the 10th and 90th percentiles. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test (**, P < 0.01; ***, P < 0.001; n.s., not significant). Source data are available for this figure: .

Article Snippet: Rabbit anti-CROCC (HPA021191; IS, 1:200; IB, 1:300; Sigma-Aldrich), mouse anti-acetylated tubulin (T6793; IS, 1:200; Sigma-Aldrich), mouse anti-α-tubulin (T6199; IS, 1:1,000; IB, 1:10,000; Sigma-Aldrich), goat anti-FLAG (ab1257; IS, 1:500; IB, 1:2,000; Abcam), rabbit anti-CEP290 (ab84870; IS, 1:100; IB, 1:200; Abcam), rabbit anti-OFD1 (ab97861; IS, 1:100; IB, 1:100; Abcam), mouse anti-FLAG (F3165; IS, 1:2,000; IB, 1:20,000; Sigma-Aldrich), rabbit anti-γ-tubulin (ab11317; IS, 1:300; Abcam), rabbit anti-centrin-2 (04-1624; IS, 1:500; Millipore), mouse anti-γ-tubulin (ab11316; IS, 1:300; Abcam), rabbit anti-CEP68 (15147-1-AP; IS, 1:100; IB, 1:500; Proteintech), rabbit anti-CEP72 (A301-297A; IS, 1:500; IB, 1:500; Bethyl), and mouse anti-GAPDH (AM4300; IB, 1:10,000; Invitrogen) antibodies were purchased from commercial suppliers.

Techniques: Expressing

Journal: The Journal of Cell Biology

Article Title: The intercentriolar fibers function as docking sites of centriolar satellites for cilia assembly

doi: 10.1083/jcb.202105065

Figure Lengend Snippet: Specific interaction of CROCC with PCM1 is essential for cilia assembly. (A) FLAG-CROCC FL and FLAG-CROCC 303–1741 were stably expressed in the CROCC KO RPE1 cells. The cells were coimmunostained with antibodies specific to CROCC (cyan) and centrin-2 (magenta). The CROCC fibers were observed with a super-resolution microscope. Scale bar, 2 μm. (B) The cells were coimmunostained with antibodies specific to acetylaged tubulin (magenta) and PCM1 (cyan). (C) Intensities of PCM1 at the centrosomes were determined. Within each box, the black center line represents the median value, the black box contains the interquartile range, and the black whiskers extend to the 10th and 90th percentiles. (D) The cells were cultured in a serum-deprived medium for 48 h, and coimmunostained with antibodies specific to acetylated tubulin (magenta) and FLAG (cyan). (E) The number of cells with cilia was counted. Graph values are expressed as mean and SEM. (B and D) Scale bars, 10 μm; inset scale bars, 2 μm. (C and E) More than 30 cells per group were counted in three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test (***, P < 0.001; n.s., not significant).

Article Snippet: Rabbit anti-CROCC (HPA021191; IS, 1:200; IB, 1:300; Sigma-Aldrich), mouse anti-acetylated tubulin (T6793; IS, 1:200; Sigma-Aldrich), mouse anti-α-tubulin (T6199; IS, 1:1,000; IB, 1:10,000; Sigma-Aldrich), goat anti-FLAG (ab1257; IS, 1:500; IB, 1:2,000; Abcam), rabbit anti-CEP290 (ab84870; IS, 1:100; IB, 1:200; Abcam), rabbit anti-OFD1 (ab97861; IS, 1:100; IB, 1:100; Abcam), mouse anti-FLAG (F3165; IS, 1:2,000; IB, 1:20,000; Sigma-Aldrich), rabbit anti-γ-tubulin (ab11317; IS, 1:300; Abcam), rabbit anti-centrin-2 (04-1624; IS, 1:500; Millipore), mouse anti-γ-tubulin (ab11316; IS, 1:300; Abcam), rabbit anti-CEP68 (15147-1-AP; IS, 1:100; IB, 1:500; Proteintech), rabbit anti-CEP72 (A301-297A; IS, 1:500; IB, 1:500; Bethyl), and mouse anti-GAPDH (AM4300; IB, 1:10,000; Invitrogen) antibodies were purchased from commercial suppliers.

Techniques: Stable Transfection, Microscopy, Cell Culture

Journal: The Journal of Cell Biology

Article Title: The intercentriolar fibers function as docking sites of centriolar satellites for cilia assembly

doi: 10.1083/jcb.202105065

Figure Lengend Snippet: Colocalization of PCM1 with subcellular CROCC. (A) The CEP250 and CROCC KO RPE1 cells were treated with 20 μM nocodazole for 2 h and subjected to coimmunostaining analysis with antibodies specific to PCM1 (cyan) and centrin-2 (magenta). (B) Intensities of PCM1 at the centrosome were determined. More than 30 cells per group were counted in three independent experiments. Within each box, the black center line represents the median value, the black box contains the interquartile range, and the black whiskers extend to the 10th and 90th percentiles. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test (***, P < 0.001; n.s., not significant). (C) The cells expressing the ectopic FLAG-CROCC protein were treated with 20 μM nocodazole for 2 h and subjected to immunoprecipitation analysis with the FLAG antibody, followed by immunoblot analyses with antibodies specific to FLAG and PCM1. (D) FLAG-CROCC FL , FLAG-CROCC 303–1741 , and FLAG-CROCC ΔR3 were stably expressed in the CROCC KO RPE1 cells. The cells were treated with 20 μM nocodazole for 2 h and coimmunostained with antibodies specific to FLAG (cyan) and PCM1 (magenta). (A and D) Scale bars, 10 μm; inset scale bar, 2 μm. Source data are available for this figure: .

Article Snippet: Rabbit anti-CROCC (HPA021191; IS, 1:200; IB, 1:300; Sigma-Aldrich), mouse anti-acetylated tubulin (T6793; IS, 1:200; Sigma-Aldrich), mouse anti-α-tubulin (T6199; IS, 1:1,000; IB, 1:10,000; Sigma-Aldrich), goat anti-FLAG (ab1257; IS, 1:500; IB, 1:2,000; Abcam), rabbit anti-CEP290 (ab84870; IS, 1:100; IB, 1:200; Abcam), rabbit anti-OFD1 (ab97861; IS, 1:100; IB, 1:100; Abcam), mouse anti-FLAG (F3165; IS, 1:2,000; IB, 1:20,000; Sigma-Aldrich), rabbit anti-γ-tubulin (ab11317; IS, 1:300; Abcam), rabbit anti-centrin-2 (04-1624; IS, 1:500; Millipore), mouse anti-γ-tubulin (ab11316; IS, 1:300; Abcam), rabbit anti-CEP68 (15147-1-AP; IS, 1:100; IB, 1:500; Proteintech), rabbit anti-CEP72 (A301-297A; IS, 1:500; IB, 1:500; Bethyl), and mouse anti-GAPDH (AM4300; IB, 1:10,000; Invitrogen) antibodies were purchased from commercial suppliers.

Techniques: Expressing, Immunoprecipitation, Western Blot, Stable Transfection

Journal: The Journal of Cell Biology

Article Title: The intercentriolar fibers function as docking sites of centriolar satellites for cilia assembly

doi: 10.1083/jcb.202105065

Figure Lengend Snippet: Generation and feature of PCM1 KO RPE1 cells. (A) The PCM1 KO RPE1 cells were coimmunostained with antibodies specific to PCM1 (cyan) and centrin-2 (magenta). (B) The PCM1 KO cells were subjected to immunoblot analyses with antibodies specific to PCM1 and GAPDH. (C) The PCM1 KO cells were subjected to immunoblot analyses with antibodies specific to PCM1, CEP290, OFD1, CEP90, CEP131, and GAPDH. (D) The PCM1 KO cells were cultured in a serum-deprived medium for 48 h and coimmunostained with antibodies specific to acetylated tubulin (magenta), along with PCM1, CEP290, OFD1, CEP131, and CEP90 (cyan). (E) Centrosome intensities of PCM1, CEP290, OFD1, CEP131, and CEP90 were determined. (F) The PCM1 KO cells were coimmunostained with antibodies specific to centrin-2 (magenta), along with CEP250 and CROCC (cyan). (G) Centrosome intensities of CEP250 and CROCC were determined. (H) The PCM1 KO cells were subjected to immunoblot analyses with antibodies specific to CEP250, CROCC, and GAPDH. (I) The FLAG-PCM1-expressing cells were coimmunostained with antibodies specific to PCM1 (cyan) and centrin-2 (magenta). (A, D, F, and I) Scale bars, 10 μm; inset scale bars, 2 μm. (E and G) More than 30 cells per group were counted in three independent experiments. Within each box, the black center line represents the median value, the black box contains the interquartile range, and the black whiskers extend to the 10th and 90th percentiles. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test (***, P < 0.001; n.s., not significant). Source data are available for this figure: .

Article Snippet: Rabbit anti-CROCC (HPA021191; IS, 1:200; IB, 1:300; Sigma-Aldrich), mouse anti-acetylated tubulin (T6793; IS, 1:200; Sigma-Aldrich), mouse anti-α-tubulin (T6199; IS, 1:1,000; IB, 1:10,000; Sigma-Aldrich), goat anti-FLAG (ab1257; IS, 1:500; IB, 1:2,000; Abcam), rabbit anti-CEP290 (ab84870; IS, 1:100; IB, 1:200; Abcam), rabbit anti-OFD1 (ab97861; IS, 1:100; IB, 1:100; Abcam), mouse anti-FLAG (F3165; IS, 1:2,000; IB, 1:20,000; Sigma-Aldrich), rabbit anti-γ-tubulin (ab11317; IS, 1:300; Abcam), rabbit anti-centrin-2 (04-1624; IS, 1:500; Millipore), mouse anti-γ-tubulin (ab11316; IS, 1:300; Abcam), rabbit anti-CEP68 (15147-1-AP; IS, 1:100; IB, 1:500; Proteintech), rabbit anti-CEP72 (A301-297A; IS, 1:500; IB, 1:500; Bethyl), and mouse anti-GAPDH (AM4300; IB, 1:10,000; Invitrogen) antibodies were purchased from commercial suppliers.

Techniques: Western Blot, Cell Culture, Expressing