Journal: Cells
Article Title: The Immune Checkpoint Protein PD-L1 Regulates Ciliogenesis and Hedgehog Signaling
doi: 10.3390/cells13121003
Figure Lengend Snippet: PD-L1 is located at the centrosome and Golgi. ( A ) Western blot analysis indicating the differential expression of PD-L1 in fibroblast and epithelial cells. ( B ) NIH3T3 cells stained with PD-L1 (green) antibody and co-stained with centrosome marker, γ-tubulin (red) (top panel), and cilium marker, acetylated-α-tubulin (red) (bottom panel). Centrosome localized PD-L1 is indicated with the white arrow. ( C ) RCTE cells stained with PD-L1 (green) antibody and co-stained with γ-tubulin (red) (top panel) and acetylated-α-tubulin (red) (bottom panel). Cells were serum starved to induce cilia growth. ( D ) RCTE cells stained with PD-L1 (green) antibody and co-stained with Golgi marker, GIANTIN (red). ( E ) Overexpression of GFP-PD-L1 (green) co-stained with Golgi marker, Giantin (red) in RCTE cells. ( F ) Overexpression of GFP-PD-L1 (green) co-stained with Golgi-associated protein Ift20 (red) in NIH3T3 cells. All cells were counterstained with DAPI (blue). Scale bars, 20 μm.
Article Snippet: Primary antibodies used in this study are listed as follows: mouse monoclonal antibodies against acetylated α-tubulin (6-11B-1, Sigma, St. Louis, MO, USA, T7451, 1:4000 used for immunofluorescence [IF]), γ-tubulin (GTU-88, Sigma, T5326, 1:1000 used for IF), α-tubulin (DM1A, sc-32293, 1:1000), Ninein (F-7, Santa Cruz, Dallas, TX, USA, sc-390540, 1:300 for IF), Cep164 (E-9, Santa Cruz, sc-515403, 1:300 used for IF), C-nap1 (E-9, Santa Cruz, sc-515403, 1:300 used for IF), Smo (E-5, Santa Cruz, sc-166685, 1:100 used for IF), Gli1 (C-1, Santa Cruz, sc-515751, 1:500 used for WB), Giantin (9B6, Abcam, Waltham, MA, USA, ab37266, 1:500 used for IF), actin (AC-15, Sigma, A1978, 1:3000 used for Western blot [WB]), GFP-tag (B-2, sc-9996, 1:1000 for WB), and Myc (9E10, sc-40, 1:1000 for WB); rabbit polyclonal antibodies against PD-L1 (Proteintech, Rosemont, IL, USA, 17952-1-AP, 1:300 used for IF, and 1:1000 used for WB), Cep135 (Abcam, ab75005, 1:300 used for IF), Ift140 (Proteintech, 17460-1-AP, 1:300 used for IF, and 1:1000 for WB), Ift20 (Proteintech, 13615-1-AP, 1:300 used for IF, and 1:1000 for WB), Rab8a (Proteintech, 55296-1-AP, 1:300 used for IF, and 1:1000 for WB), BBS5 (Proteintech, 14569-1-AP, 1:300 used for IF, and 1:1000 for WB), ARL13B (Proteintech, 17711-1-AP, 1:2000 for IF), GFP-tag (Proteintech, 50430-2-AP, 1:1000 for WB) and Myc-Tag (CST, 71D10, 1:1000 used for WB); goat polyclonal antibody against polycystin 2 (E-20, Santa Cruz, sc-10377, 1:500 for WB).
Techniques: Western Blot, Expressing, Staining, Marker, Over Expression
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