10f  (ATCC)

Journal: Cell Discovery

Article Title: Architecture, substructures, and dynamic assembly of STRIPAK complexes in Hippo signaling

doi: 10.1038/s41421-018-0077-3

Figure Lengend Snippet: a Schematic representation of the domain organization of the core STRIPAK components. CC, coiled-coil domain; WD40, WD40 repeat; Kinase, kinase domain; SARAH, Sav-RASSF-Hpo domain; NTD, N-terminal domain; CTD, C-terminal domain; FHA, forkhead-associated domain; TM, transmembrane domain. b Effect of the knockdown of STRIPAK components on the activation of Hippo signaling in HGC-27 cells. n.c., negative control (control siRNA). c Real-time PCR analysis of the expression levels of CTGF in HGC-27 cells transfected with the indicated siRNAs. Bar graphs represent the means ± SD. Experiments were repeated three times. Unpaired t tests were used to compare the difference between the two groups. *Significant relative to control, p < 0.05, ** p < 0.01, *** p < 0.001. d MBP pulldown analysis of the PP2A holoenzyme containing STRN3 as its B′′′ subunit. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with Coomassie brilliant blue (CBB). e MBP pulldown analysis of the interactions between core STRIPAK components and the scaffold protein STRN3. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. f MBP pulldown analysis of the interactions between core STRIPAK components and the MST2 protein kinase. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. g Model of the overall assembly of the STRIPAK complex

Article Snippet: An antibody specific for Flag (F3165) was obtained from Sigma; antibodies to HA (rabbit, #3724), MST1 (rabbit, #3682), MOB1 (#13730), p-MOB1 (T35, #8699), p-YAP (S127, #13008), MST1 (#3682), and p-MST1 (T183)/p-MST2 (T180) (#3681) were from Cell Signaling Technology; antibodies to YAP (sc-101199), SLMAP (sc-100957), HA (mouse, sc-7392), MST1 (mouse, sc-515051), and MST2 (sc-130405) were from Santa Cruz; an antibody to STRN3 (#PA5-31368) was from Invitrogen; and antibodies to SIKE1 (ab121860) and STRIP1 (ab199851) were from Abcam.

Techniques: Activation Assay, Negative Control, Real-time Polymerase Chain Reaction, Expressing, Transfection, SDS Page, Staining

Journal: Cell Discovery

Article Title: Architecture, substructures, and dynamic assembly of STRIPAK complexes in Hippo signaling

doi: 10.1038/s41421-018-0077-3

Figure Lengend Snippet: a MBP-pulldown-based determination of the domains of STRN3 most responsible for binding STRIP1. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. b MBP-pulldown-based determination of the STRN3-binding domains of STRIP1. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. HS, His-SUMO tag. c ITC analysis of the interaction between STRN3 (64–145) and STRIP1 (765–837). d Co-immunoprecipitation and immunoblot analysis of STRN3 and STRIP1 in HEK293FT cells. Δ796–837, deletion of residues 796–837; Δ64–145, deletion of residues 64–145; IP, immunoprecipitation. e MBP-pulldown-based determination of the domains of STRN3 most responsible for binding SIKE1. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. f MBP-pulldown-based determination of the STRN3-binding domain of SIKE1. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. g Co-immunoprecipitation and immunoblot analysis of STRN3 and SIKE1 in HEK293FT cells. Δ64–190, deletion of residues 64–190; Δ72–121, deletion of residues 72–121. h Model of STRN3-STRIP1 and STRN3-SIKE1 assembly in the STRIPAK complex. CTT, C-terminal tail

Article Snippet: An antibody specific for Flag (F3165) was obtained from Sigma; antibodies to HA (rabbit, #3724), MST1 (rabbit, #3682), MOB1 (#13730), p-MOB1 (T35, #8699), p-YAP (S127, #13008), MST1 (#3682), and p-MST1 (T183)/p-MST2 (T180) (#3681) were from Cell Signaling Technology; antibodies to YAP (sc-101199), SLMAP (sc-100957), HA (mouse, sc-7392), MST1 (mouse, sc-515051), and MST2 (sc-130405) were from Santa Cruz; an antibody to STRN3 (#PA5-31368) was from Invitrogen; and antibodies to SIKE1 (ab121860) and STRIP1 (ab199851) were from Abcam.

Techniques: Binding Assay, SDS Page, Staining, Immunoprecipitation, Western Blot

Journal: Cell Discovery

Article Title: Architecture, substructures, and dynamic assembly of STRIPAK complexes in Hippo signaling

doi: 10.1038/s41421-018-0077-3

Figure Lengend Snippet: a Cartoon views of the overall structure of apo SIKE1 CC2. Four chains of SIKE1 CC2 are colored cyan (chain A), palecyan (chain B), blue (chain A′), and lightblue (chain B′), respectively. b Interactions at the dimeric interface of SIKE1 CC2. c Interactions at the tetrameric interface of SIKE1 CC2. d Cartoon views of the overall structure of SIKE1 CC2-STRN3 complex. The two SIKE1 CC2 molecules are colored cyan (chain A) and palecyan (chain B), respectively, and the STRN3 peptides are colored yellow. e, f Zoomed-in views of SIKE1 and STRN3 interactions at the complex interface. The red dash lines represent hydrogen bonds or salt bridges. The interface residues are labeled and highlighted by the stick model

Article Snippet: An antibody specific for Flag (F3165) was obtained from Sigma; antibodies to HA (rabbit, #3724), MST1 (rabbit, #3682), MOB1 (#13730), p-MOB1 (T35, #8699), p-YAP (S127, #13008), MST1 (#3682), and p-MST1 (T183)/p-MST2 (T180) (#3681) were from Cell Signaling Technology; antibodies to YAP (sc-101199), SLMAP (sc-100957), HA (mouse, sc-7392), MST1 (mouse, sc-515051), and MST2 (sc-130405) were from Santa Cruz; an antibody to STRN3 (#PA5-31368) was from Invitrogen; and antibodies to SIKE1 (ab121860) and STRIP1 (ab199851) were from Abcam.

Techniques: Labeling

Journal: Cell Discovery

Article Title: Architecture, substructures, and dynamic assembly of STRIPAK complexes in Hippo signaling

doi: 10.1038/s41421-018-0077-3

Figure Lengend Snippet: a Overall structural comparison of apo SIKE1 CC2 with SIKE1 CC2-STRN3 complex. b Structure-based explanation of the 2:1 stoichiometry in SIKE1 CC2-STRN3 complex. c – e Co-immunoprecipitation and immunoblot analysis of the interactions between wildtype or mutant forms of SIKE1 and STRN3 in lysates of HEK293FT cells transfected with the indicated plasmids. 4M, E96A/R109A/A100D/M105E; 4D, L90D/L94D/H97D/L101D

Article Snippet: An antibody specific for Flag (F3165) was obtained from Sigma; antibodies to HA (rabbit, #3724), MST1 (rabbit, #3682), MOB1 (#13730), p-MOB1 (T35, #8699), p-YAP (S127, #13008), MST1 (#3682), and p-MST1 (T183)/p-MST2 (T180) (#3681) were from Cell Signaling Technology; antibodies to YAP (sc-101199), SLMAP (sc-100957), HA (mouse, sc-7392), MST1 (mouse, sc-515051), and MST2 (sc-130405) were from Santa Cruz; an antibody to STRN3 (#PA5-31368) was from Invitrogen; and antibodies to SIKE1 (ab121860) and STRIP1 (ab199851) were from Abcam.

Techniques: Immunoprecipitation, Western Blot, Mutagenesis, Transfection

Journal: Cell Discovery

Article Title: Architecture, substructures, and dynamic assembly of STRIPAK complexes in Hippo signaling

doi: 10.1038/s41421-018-0077-3

Figure Lengend Snippet: a GST-pulldown-based determination of the MST2-binding domain of STRN3. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. b Co-immunoprecipitation and immunoblot analysis of STRN3 and MST2 in HEK293FT cells. Δ64–145, deletion of residues 64–145; K56R, a form of MST2 displaying no kinase activity. c MBP-pulldown-based determination of the STRN3-binding domain of MST2. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. d MBP pulldown analysis of the interaction between STRN3 and the wildtype or mutant form of MST2. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. e Model of STRN3-MST2 assembly in the STRIPAK complex. Kinase, kinase domain; DD, dimerization domain, which is the SARAH domain in MST2 and DD in MST4

Article Snippet: An antibody specific for Flag (F3165) was obtained from Sigma; antibodies to HA (rabbit, #3724), MST1 (rabbit, #3682), MOB1 (#13730), p-MOB1 (T35, #8699), p-YAP (S127, #13008), MST1 (#3682), and p-MST1 (T183)/p-MST2 (T180) (#3681) were from Cell Signaling Technology; antibodies to YAP (sc-101199), SLMAP (sc-100957), HA (mouse, sc-7392), MST1 (mouse, sc-515051), and MST2 (sc-130405) were from Santa Cruz; an antibody to STRN3 (#PA5-31368) was from Invitrogen; and antibodies to SIKE1 (ab121860) and STRIP1 (ab199851) were from Abcam.

Techniques: Binding Assay, SDS Page, Staining, Immunoprecipitation, Western Blot, Activity Assay, Mutagenesis

Journal: Cell Discovery

Article Title: Architecture, substructures, and dynamic assembly of STRIPAK complexes in Hippo signaling

doi: 10.1038/s41421-018-0077-3

Figure Lengend Snippet: a MBP pulldown analysis of the interaction between SIKE1/FGFR1OP2 and SLMAP or STRIP1. FGFR1OP2, a homolog of SIKE1. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. b MBP pulldown analysis of the interaction between SLMAP and STRN3 in the absence or presence of SIKE1. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. c MBP-pulldown-based determination of the SLMAP-binding domain of SIKE1. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. d MBP-pulldown-based determination of the SIKE1-binding domain of SLMAP. The input and output samples were loaded on an SDS-PAGE gel followed by staining this gel with CBB. e Co-immunoprecipitation and immunoblot analysis of SLMAP and SIKE1 in HEK293FT cells. Δ161–230, deletion of residues 161–230; Δ1–48, deletion of residues 1–48. f Model of SLMAP-SIKE1 assembly in the STRIPAK complex

Article Snippet: An antibody specific for Flag (F3165) was obtained from Sigma; antibodies to HA (rabbit, #3724), MST1 (rabbit, #3682), MOB1 (#13730), p-MOB1 (T35, #8699), p-YAP (S127, #13008), MST1 (#3682), and p-MST1 (T183)/p-MST2 (T180) (#3681) were from Cell Signaling Technology; antibodies to YAP (sc-101199), SLMAP (sc-100957), HA (mouse, sc-7392), MST1 (mouse, sc-515051), and MST2 (sc-130405) were from Santa Cruz; an antibody to STRN3 (#PA5-31368) was from Invitrogen; and antibodies to SIKE1 (ab121860) and STRIP1 (ab199851) were from Abcam.

Techniques: SDS Page, Staining, Binding Assay, Immunoprecipitation, Western Blot

Journal: Cell Discovery

Article Title: Architecture, substructures, and dynamic assembly of STRIPAK complexes in Hippo signaling

doi: 10.1038/s41421-018-0077-3

Figure Lengend Snippet: a Co-immunoprecipitation and immunoblot analysis of the endogenous interactions between core STRIPAK components and STRN3 in HEK293FT cells at two different cell densities. b Co-immunoprecipitation and immunoblot analysis of the endogenous interactions between core STRIPAK components and MST1 in HEK293FT cells at two different cell densities. c Confocal microscope analysis of the colocalization of MST2 or STRIP1 with STRN3 in HEK293FT cells at two different cell densities. The rate of colocalization was quantified and shown (right). d Model of the dynamic assembly of the STRIPAK complex induced by different cell densities

Article Snippet: An antibody specific for Flag (F3165) was obtained from Sigma; antibodies to HA (rabbit, #3724), MST1 (rabbit, #3682), MOB1 (#13730), p-MOB1 (T35, #8699), p-YAP (S127, #13008), MST1 (#3682), and p-MST1 (T183)/p-MST2 (T180) (#3681) were from Cell Signaling Technology; antibodies to YAP (sc-101199), SLMAP (sc-100957), HA (mouse, sc-7392), MST1 (mouse, sc-515051), and MST2 (sc-130405) were from Santa Cruz; an antibody to STRN3 (#PA5-31368) was from Invitrogen; and antibodies to SIKE1 (ab121860) and STRIP1 (ab199851) were from Abcam.

Techniques: Immunoprecipitation, Western Blot, Microscopy