ift88 fusion protein ag4980  (Proteintech)

Journal: Cell reports

Article Title: Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis.

doi: 10.1016/j.celrep.2020.107932

Figure Lengend Snippet: Figure 4. IFT88 and IFT20 Are Physically Associated to YAP1, and IFT88 Modulates YAP1 Activity (A) Co-IP experiment using HeLa cells transfected with IFT88-GFP, HA-Amotl1, and YAP1-Myc (l.e., long exposure; s.e., short exposure). Co-IP experiment using HEK293 cells transfected with Flag-Amotl1 and IFT20-GFP. Endogenous levels of Yap1 are monitored. (B) Schematic representation of the IFT88 auxin-inducible degron (AID) system. (C) Western blot analysis of IFT88 AID DLD-1 cells after 2-h auxin treatment. (C0) Western blot analysis of IFT88 and YAP1 degradation after auxin treatment (0, 0.5, and 2 h). (D) Graph shows the increase in normalized YAP1 nuclear signal in cells treated with auxin (6 h) (Mann-Whitney, p value < 0.0001) (controls: 2 replicates, n = 204 cells; Auxin 2 h: 2 replicates, n = 261 cells). (E) Graph shows the increase in YAP/WWTR1 (TAZ) nuclear signal in IFT88-siRNA (48 h)-treated MDCK cells (n = 5 replicates, average cell number analyzed for each condition = 382; t test, p value, 0.004). Box and whiskers (5th–95th percentile). Outliers are represented as red dots (NT-siRNA) or blue squares (IFT88- siRNA).

Article Snippet: We performed IPs using GFP-Trap (ChromoTek) agarose beads in two conditions: Control IP (YAP1-Myc (Boin et al., 2014), pEGFPC1 and HA-Amotl1 (Ragni et al., 2017)) and IFT88 IP (YAP1-Myc, IFT88-GFP (He et al., 2014) and HA-Amotl1).

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Transfection, Western Blot, MANN-WHITNEY

Journal: Methods in cell biology

Article Title: Analysis of primary cilia in renal tissue and cells

doi: 10.1016/bs.mcb.2019.04.008

Figure Lengend Snippet: Markers of primary cilia and renal tubules.

Article Snippet: Ciliary markers include acetylated α-tubulin, which labels the stable microtubules of the ciliary axoneme ( Pazour et al., 2002 ; Qian et al., 2005 ; Schraml et al., 2009 ; Verghese et al., 2009 ), ARL13B, a small ciliary GTPase of Arf/Arl family that localizes to the ciliary membrane ( Caspary et al., 2007 ), and IFT88, a component of the IFT-B complex that localizes punctately throughout the axoneme ( Taulman et al., 2001 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody or lectin Company Localization References Acetylated α-tubulin Sigma-Aldrich Axoneme Pazour, San Agustin, Follit, Rosenbaum, and Witman (2002) , Qian et al. (2005) , Schraml et al. (2009) , and Verghese et al. (2009) ARL13b Proteintech Ciliary membrane Caspary, Larkins, and Anderson (2007) and Ma et al. (2013) IFT88 Proteintech Punctate along axoneme Taulman, Haycraft, Balkovetz, and Yoder (2001) Aquaporin-2 Santa-Cruz Biotechnology Collecting duct Verghese et al. (2009) Tamm-Horsfall Protein (THP) Santa-Cruz Biotechnology Loop of Henle Tran et al. (2014) Lotus tetragonolobus lectin (LTL) Vector Laboratories Proximal tubule Sohara et al. (2008) and Verghese et al. (2009) Dolichos biflorus agglutinin (DBA) Vector Laboratories Collecting duct Ma et al. (2013) , Sohara et al. (2008) , and Verghese et al. (2009) Open in a separate window Markers of primary cilia and renal tubules.

Techniques: Plasmid Preparation

Journal: Methods in cell biology

Article Title: Analysis of primary cilia in renal tissue and cells

doi: 10.1016/bs.mcb.2019.04.008

Figure Lengend Snippet: Markers of primary cilia.

Article Snippet: Ciliary markers include acetylated α-tubulin, which labels the stable microtubules of the ciliary axoneme ( Pazour et al., 2002 ; Qian et al., 2005 ; Schraml et al., 2009 ; Verghese et al., 2009 ), ARL13B, a small ciliary GTPase of Arf/Arl family that localizes to the ciliary membrane ( Caspary et al., 2007 ), and IFT88, a component of the IFT-B complex that localizes punctately throughout the axoneme ( Taulman et al., 2001 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody or lectin Company Localization References Acetylated α-tubulin Sigma-Aldrich Axoneme Pazour, San Agustin, Follit, Rosenbaum, and Witman (2002) , Qian et al. (2005) , Schraml et al. (2009) , and Verghese et al. (2009) ARL13b Proteintech Ciliary membrane Caspary, Larkins, and Anderson (2007) and Ma et al. (2013) IFT88 Proteintech Punctate along axoneme Taulman, Haycraft, Balkovetz, and Yoder (2001) Aquaporin-2 Santa-Cruz Biotechnology Collecting duct Verghese et al. (2009) Tamm-Horsfall Protein (THP) Santa-Cruz Biotechnology Loop of Henle Tran et al. (2014) Lotus tetragonolobus lectin (LTL) Vector Laboratories Proximal tubule Sohara et al. (2008) and Verghese et al. (2009) Dolichos biflorus agglutinin (DBA) Vector Laboratories Collecting duct Ma et al. (2013) , Sohara et al. (2008) , and Verghese et al. (2009) Open in a separate window Markers of primary cilia and renal tubules.

Techniques:

Journal: Methods in cell biology

Article Title: Analysis of primary cilia in renal tissue and cells

doi: 10.1016/bs.mcb.2019.04.008

Figure Lengend Snippet: Markers of primary cilia and renal tubules.

Article Snippet: The balance between cilia assembly and disassembly regulates cilia length ( Mirvis, Stearns, & James Nelson, 2018 ; Spalluto, Wilson, & Hearn, 2013 ). table ft1 table-wrap mode="anchored" t5 caption a7 Cell line Description Source References M-1 Murine cortical collecting duct epithelial cells ATCC Stoos et al. (1991) IMCD-3 Murine inner medullary collecting duct epithelial cells ATCC Rauchman et al. (1993) LLC-PK1 Porcine proximal tubule epithelial cells ATCC Nielsen et al. (1998) MDCK Madin-Darby canine kidney epithelial cells ATCC Gaush et al. (1966) NHK/ADPKD Primary cortical epithelial cells from normal human kidney (NHK) or Autosomal Dominant PKD (ADPKD) KUMC Graham et al. (1977) and Reif et al. (2011) Open in a separate window Immortalized and primary renal epithelial cells. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Company Cilia structure References Acetylated-α-tubulin Sigma-Aldrich Axoneme Ishikawa et al. (2012) , Seixas et al. (2015) , Silva et al. (2018) , and Yu, Sharma, Skowronek, and Erdmann (2016) γ-tubulin Sigma-Aldrich Centrioles Breslow et al. (2013) Pericentrin Covance Centrioles Ishikawa et al. (2012) ARL13B Proteintech Ciliary membrane Seixas et al. (2015) INPP5E Proteintech Ciliary membrane Plotnikova et al. (2015) IFT52 Proteintech Axoneme Silva et al. (2018) IFT81 Proteintech Axoneme Silva et al. (2018) IFT88 Proteintech Axoneme Silva et al. (2018) IFT140 Proteintech Axoneme Silva et al. (2018) BBS2 Proteintech Axoneme Silva et al. (2018) BBS5 Proteintech Axoneme Silva et al. (2018) Open in a separate window Markers of primary cilia.

Techniques: Plasmid Preparation

Journal: Methods in cell biology

Article Title: Analysis of primary cilia in renal tissue and cells

doi: 10.1016/bs.mcb.2019.04.008

Figure Lengend Snippet: Markers of primary cilia.

Article Snippet: The balance between cilia assembly and disassembly regulates cilia length ( Mirvis, Stearns, & James Nelson, 2018 ; Spalluto, Wilson, & Hearn, 2013 ). table ft1 table-wrap mode="anchored" t5 caption a7 Cell line Description Source References M-1 Murine cortical collecting duct epithelial cells ATCC Stoos et al. (1991) IMCD-3 Murine inner medullary collecting duct epithelial cells ATCC Rauchman et al. (1993) LLC-PK1 Porcine proximal tubule epithelial cells ATCC Nielsen et al. (1998) MDCK Madin-Darby canine kidney epithelial cells ATCC Gaush et al. (1966) NHK/ADPKD Primary cortical epithelial cells from normal human kidney (NHK) or Autosomal Dominant PKD (ADPKD) KUMC Graham et al. (1977) and Reif et al. (2011) Open in a separate window Immortalized and primary renal epithelial cells. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Company Cilia structure References Acetylated-α-tubulin Sigma-Aldrich Axoneme Ishikawa et al. (2012) , Seixas et al. (2015) , Silva et al. (2018) , and Yu, Sharma, Skowronek, and Erdmann (2016) γ-tubulin Sigma-Aldrich Centrioles Breslow et al. (2013) Pericentrin Covance Centrioles Ishikawa et al. (2012) ARL13B Proteintech Ciliary membrane Seixas et al. (2015) INPP5E Proteintech Ciliary membrane Plotnikova et al. (2015) IFT52 Proteintech Axoneme Silva et al. (2018) IFT81 Proteintech Axoneme Silva et al. (2018) IFT88 Proteintech Axoneme Silva et al. (2018) IFT140 Proteintech Axoneme Silva et al. (2018) BBS2 Proteintech Axoneme Silva et al. (2018) BBS5 Proteintech Axoneme Silva et al. (2018) Open in a separate window Markers of primary cilia.

Techniques: