Journal: Human Molecular Genetics

Article Title: A meckelin–filamin A interaction mediates ciliogenesis

doi: 10.1093/hmg/ddr557

Figure Lengend Snippet: Loss of meckelin or filamin A causes deregulation of Wnt signalling. (A) TopFlash assays to measure levels of activated β-catenin, and hence canonical Wnt signalling, following co-transfection of immortalized normal control (control fibs, blue bars) and MKS3-mutated dermal fibroblasts (MKS3 fibs, red bars; see Fig. 3C) with reporter constructs and empty vector, wild-type HA-meckelin, p.F919del mutant HA-meckelin constructs and c myc-filamin A, as indicated. The empty vector results combine the data from transfections with pCMV-HA and pCMV-c myc. Activity is expressed as ratios of luciferase reporter construct expression, normalized for loading by measurement of a Renilla construct expression. The responses are shown to 0.5 × L cell control conditioned media (control), and conditioned media containing expressed Wnt3A and/or Wnt5A. Values shown are means of at least four independent replicates, with error bars indicating SEM. Statistical significance of pair-wise comparisons are shown (*P < 0.01 and **P < 0.001, Student's t-test). (B) TopFlash reporter assays as in (A) for control fibroblasts (control fibs, blue bars) and FLNA-mutated fibroblasts (FLNA fibs, green bars). Values shown are means of three independent replicates. Statistical significance of pair-wise comparisons are shown (*P < 0.05, error bars = SEM, **P < 0.01, Student's t-test).

Article Snippet: For luciferase assays of canonical Wnt activity, we grew fibroblasts in 12-well plates and co-transfected with 0.5 μg of Topflash firefly luciferase construct (or Fopflash, as a negative control), 0.5 μg of expression constructs (pCMV HA-meckelin, pCMV c myc-filamin A or empty pCMV-HA/pCMV c myc vector) and 0.1 μg of pRL-TK (Promega Corp., WI, USA; Renilla luciferase construct used as an internal control reporter).

Techniques: Cotransfection, Control, Construct, Plasmid Preparation, Mutagenesis, Transfection, Activity Assay, Luciferase, Expressing

Journal: Biomolecules

Article Title: OTUD7B Activates Wnt Signaling Pathway through the Interaction with LEF1

doi: 10.3390/biom13061001

Figure Lengend Snippet: OTUD7B interacts with LEF1 and activates Wnt signaling. ( A ) HEK293T cells were transiently transfected with MYC-LEF1 and SFB-tagged DUBs (USP26, USP36, USP42, and OTUD7B) and subjected to pulldown using S-protein beads. The pulled-down DUBs and DUB-bound LEF1 were detected via immunoblotting using anti-FLAG and anti-MYC antibodies, respectively. ( B ) HEK293T cells were transiently transfected with MYC-LEF1 and SFB-OTUD7B and subjected to immunoprecipitation using MYC antibody-conjugated agarose beads. The pulled-down LEF1 and bound OTUD7B were detected via immunoblotting using anti-MYC and FLAG antibodies, respectively. ( C ) HEK293T cells were co-transfected with TOPflash (LEF1/β-catenin-responsive luciferase vector) or its mutant FOPflash, along with Renilla luciferase and SFB-USP36 or SFB-OTUD7B plasmids. Firefly luciferase activity from TOPflash and FOPflash was normalized to Renilla luciferase activity. The normalized TOPflash luminescence levels were further normalized to each normalized FOPflash luminescence. SFB-GFP was used as a negative control. ( D ) HEK293T cells were co-transfected with TOPflash or FOPflash, along with Renilla luciferase, and control siRNA or siRNA for OTUD7B. Left panel: the cells were subjected to lysis and immunoblotting was conducted using OTUD7B antibody to confirm the knockdown of endogenous OTUD7B. Right panel: Firefly luciferase activity from TOPflash and FOPflash was normalized to Renilla luciferase activity. The normalized TOPflash luminescence levels were further normalized to each normalized FOPflash luminescence. Control siRNA-transfected group was used as a negative control. Error bars represent the standard error of the mean (S.E.M.). Statistical significance was determined using an unpaired t -test.

Article Snippet: HA-ubiquitin, TOPflash, and FOPflash vectors were obtained from Addgene (Item #17608, #12456, and #12457, respectively; Watertown, MA, USA).

Techniques: Transfection, Western Blot, Immunoprecipitation, Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Negative Control, Control, Lysis, Knockdown