active q67l pgfp rab8a  (Addgene inc)

Journal: EMBO Reports

Article Title: TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution

doi: 10.15252/embr.201948901

Figure Lengend Snippet: RPE1 cells were transfected with siRNAs as indicated, followed by transfection with wild‐type pGFP‐Rab8a (WT‐Rab8), constitutively active pGFP‐Rab8a (Q67L) (CA‐Rab8), or DN Rab8 dominant‐negative pGFP Rab8a (T22N), incubated in serum‐starved media for 12 h, and immunostained for ARL13B (red), GFP‐Rab8 (green), and DAPI (blue). Scale bar, 10 μm. Quantification of the percentage of ciliated cells shown in (A). Data represent mean ± SD ( n = 3 experiments), and 200 GFP‐positive cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. (Upper panel) Cells were transfected as indicated and the cell lysates were incubated with purified proteins, including GST or GST‐JCF1 (RBD). The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Intensity of the bands was quantified by ImageJ software. The amount of GTP‐Rab8 was normalized to the control level. Bar graph represents mean ± SD ( n = 3 experiments). * P < 0.05, Student's t ‐test. Cells were transfected as shown in (A), and cell lysates were incubated with purified GST‐JCF1 (RBD) fusion protein. The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Cells were transfected as shown in (A), and cell lysates were incubated with purified GST protein. The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blot with Rab8 antibody. Source data are available online for this figure.

Article Snippet: Human wild‐type pGFP‐Rab8A (Plasmid #24898), human constitutively active (Q67L) pGFP‐Rab8A (Plasmid #24900), and human dominant‐negative (T22N) pGFP‐Rab8A (Plasmid #24899) were obtained from Addgene.

Techniques: Transfection, Dominant Negative Mutation, Incubation, Purification, Western Blot, Software, Control

Journal: EMBO Reports

Article Title: TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution

doi: 10.15252/embr.201948901

Figure Lengend Snippet: RPE1 cells were transfected with siRNAs as indicated, followed by 24‐h incubation in serum‐starved media. Cells were then and subjected to fractionation, and Western blot for IFT20, the Golgi marker GM130, membrane marker UBXD8, and nuclear marker CREB. Efficiency of IFT20 knockdown by Western blot. Cells were transfected with siRNAs as indicated, followed by incubation in serum‐starved media for 24 h, and immunostained for ARL13B (red). Scale bar, 10 μm. The bar graph represents the quantification of the percentage of ciliated cells. Data represent mean ± SD ( n = 3 experiments), and 250 cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. Cells were transfected as shown in (C), and immunostained for Rab8 and γ‐tubulin, followed by quantification of the percentage of cells with Rab8 localized to the centriole. Data represent average ( n = 2 experiments). Cells were transfected with siRNAs as indicated, followed by transfection with Flag‐IFT20, incubated in serum‐starvation media for 12 h, and immunostained for ARL13B. Representative fluorescent images of Flag‐IFT20 (green), ARL13B (red), and DAPI (blue) are shown. Scale bar, 10 μm. Quantification of the percentage of ciliated cells with both the Flag‐IFT20 and ARL13B localized in the cilium. Data represent mean ± SD ( n = 3 experiments), and 150 Flag‐positive cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. Cells were transfected with siRNAs as indicated, followed by further transfection with CA‐Rab8, incubated in serum‐starvation media for 12 h, and immunostained for acetylated tubulin. Representative fluorescent images of GFP‐Rab8 Q67L (green), acetylated tubulin (red), and DNA (blue) are shown. Scale bar, 10 μm. Quantification of the percentage of GFP‐positive ciliated cells (only those cilia having both GFP‐Rab8 and acetylated tubulin on cilium were considered for quantification). Data represent mean ± SD ( n = 3 experiments), and 200 GFP‐positive cells were scored per condition per experiment, * P < 0.05, Student's t ‐test. Source data are available online for this figure.

Article Snippet: Human wild‐type pGFP‐Rab8A (Plasmid #24898), human constitutively active (Q67L) pGFP‐Rab8A (Plasmid #24900), and human dominant‐negative (T22N) pGFP‐Rab8A (Plasmid #24899) were obtained from Addgene.

Techniques: Transfection, Incubation, Fractionation, Western Blot, Marker, Membrane, Knockdown

Journal: EMBO Reports

Article Title: TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution

doi: 10.15252/embr.201948901

Figure Lengend Snippet: A Efficiency of Rab8a depletion confirmed by Western blot in RPE1 cells. B RPE1 cells were transfected by siRNAs as indicated, followed by incubation in serum‐starvation media for 24 h, and immunostained for ARL13B (red) and γ‐tubulin (green). Scale bar, 10 μm. C Quantification of the percentage of ciliated cells shown in (B). Data represent mean ± SD ( n = 3 experiments), and 250 GFP‐positive cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. D Cells were transfected by siRNA as indicated followed by incubation in serum‐starvation media for 24 h, and immunostained for EHD1 (red) and γ‐tubulin (green). Scale bar, 10 μm. E Quantification of the percentage of cells with EHD1 in cilium or in the distal end of basal body as shown in (D). Data represent mean ± SD ( n = 3 experiments), and 150 cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. F Cells were transfected by siRNAs as indicated, followed by incubation in serum‐starvation media for 24 h, and immunostained for IFT20 (red) and γ‐tubulin (green). Scale bar, 10 μm. G Quantification of the percentage of IFT20 fluorescent intensity at the centriole shown in (F). Data represent mean ± SD ( n = 3 experiments), and 150 were scored per condition per experiment; * P < 0.05, Student's t ‐test. H–J Cells were transfected by siRNAs as indicated, followed by transfection with GFP‐Rab8 WT, GFP‐Rab8 Q67L, or GFP‐Rab8 T22N, and further incubated in serum‐starvation media for 12 h. Cell lysate was subjected to immunoprecipitation with anti‐GFP antibody, followed by Western blot with antibody against GFP. Source data are available online for this figure.

Article Snippet: Human wild‐type pGFP‐Rab8A (Plasmid #24898), human constitutively active (Q67L) pGFP‐Rab8A (Plasmid #24900), and human dominant‐negative (T22N) pGFP‐Rab8A (Plasmid #24899) were obtained from Addgene.

Techniques: Western Blot, Transfection, Incubation, Immunoprecipitation