rabbit anti frmd6  (ABclonal Biotechnology)

Journal: Frontiers in Cellular Neuroscience

Article Title: Willin/FRMD6 Influences Mechanical Phenotype and Neuronal Differentiation in Mammalian Cells by Regulating ERK1/2 Activity

doi: 10.3389/fncel.2020.552213

Figure Lengend Snippet: Knock-down of Willin/FRMD6 affects proliferation, migration, morphology, and force exertion of SH-SY5Y cells. (A) Quantitative Western blot analysis of Willin/FRMD6 expression in control ( shScr ) and Willin/FRMD6 knock-down ( shWillin ) SH-SY5Y cells. Means were calculated from three independent experiments. Error bars represent SEM. GAPDH serves as a loading control of the lysates. (B) qPCR analysis of Willin/FRMD6 mRNA expression in shScr and shWillin cells. Means and SEM (error bars) were calculated from two independent experiments, each of which was conducted in triplicates. (C) Growth curve of shScr and shWillin cells. Means (horizontal lines) and SEM (error bars) were calculated from three independent experiments, each of which was conducted in triplicates. (D) Assessment of migration of shScr and shWillin cells in Boyden chambers after 24 h. Means (horizontal lines) and SEM (error bars) were calculated from two independent experiments, each of which was conducted in triplicates. (E) Phase-contrast images (upper row), ERISM displacement maps (middle row), and Fourier-filtered ERISM maps (lower row) of representative shScr (left column) and shWillin (right column) cell. Scale bars: 25 μm. Comparison of (F) volume by which cells indent into the ERISM substrate, (G) cell area, and (H) cell elongation of shScr and shWillin cells. Each data point represents the measured value for one cell taken from four (F) and two (G,H) independent experiments, respectively, lines indicate means, error bars SEM. Groups were compared using Student’s t -test; *** p ≤ 0.001.

Article Snippet: Membranes were blocked in either 5% skimmed dried milk in TBS-T or 5% BSA in TBS-T for 1 h at room temperature, before being incubated with primary antibody diluted in blocking solution overnight at 4°C [FRMD6: Cell Signaling Technology, 14688, 1:100; Phospho-p44/42 MAPK: Cell Signaling Technology, 4370, 1:4,000; p44/42 MAPK (ERK1/2): Cell Signaling Technology, 9107, 1:2,000; NeuroD1: Santa Cruz Biotechnology, sc-1084, 1:100; Vinculin: Novus Biologicals, NBP2-41274, 1:500; β-actin: Sigma–Aldrich, A1978, 1:20,000; GAPDH: Sigma–Aldrich, G8795, 1:20,000].

Techniques: Knockdown, Migration, Western Blot, Expressing, Control, Comparison

Journal: Frontiers in Cellular Neuroscience

Article Title: Willin/FRMD6 Influences Mechanical Phenotype and Neuronal Differentiation in Mammalian Cells by Regulating ERK1/2 Activity

doi: 10.3389/fncel.2020.552213

Figure Lengend Snippet: Knockdown of Willin/FRMD6 in SH-SY5Y cells activates the ERK1/2 pathway and elicits differentiation into a neuronal phenotype by activation of the transcriptional factor NeuroD1. Quantitative Western blot analysis of the levels of pERK1/2 relative to ERK1/2 (A) in control ( shScr ) and Willin/FRMD6 knock-down ( shWillin ) SH-SY5Y cells, (B) in control ( Vector ) and Willin/FRMD6 overexpressing ( Willin ) SH-SY5Y cells, (C) in SH-SY5Y wild type cells treated with either DMSO or retinoic acid (RA) and (D) in SH-SY5Y wild type cells treated with either DMSO or U0126. Means in (A) to (D) were calculated from three technical repeats. Error bars represent SEM. Comparison of (E) percentage of fine extension-bearing cells and (F) volume by which cells deform the ERISM substrate (each data point represents the value measured for one cell) for shScr cells treated with DMSO and shWillin cells treated with either DMSO or U0126 for 24 h. In (E) means were calculated from two independent experiments conducted in triplicate. In (F) each data point represents the value measured for one cell. Lines indicate means, error bars SEM. (G) Percentage of differentiating shScr and shWillin cells quantified from bright-field images of cells treated with 10 μM RA along with either DMSO or 10 μM U0126 for 4 days. Means and SEM (error bars) were calculated from two independent experiments, each of which was conducted in triplicates. Quantification of the levels of NeuroD1 in shScr and shWillin cells from (H) Western blot analysis and (I) qPCR analysis. Means in (H) and (I) were calculated from two independent experiments conducted in triplicate. Error bars represent SEM. (J) Immunofluorescent imaging of shScr (upper row) and shWillin (lower row) cells for NeuroD1 (left column) and DNA (right column). Scale bars: 40 μm. (K) Quantification of extension bearing cells from bright-field images for shScr cells treated with 10 μM RA for 1, 4, and 7 days. Means and SEM (error bars) were calculated from two independent experiments, each of which was conducted in triplicates. (L) Development of the volume by which cells indent into the ERISM substrate over 1 week during RA-initiated (10 μM) neuronal differentiation of shScr cells. Data points represent means measured for n i individual cells; shaded areas represent SEM (Day 1: n shScr = 16, n shScrRA = 31, n shWillin = 22; Day 3: n shScr = 21, n shScrRA = 17, n shWillin = 14; Day 6: n shScr = 12, n shScrRA = 16, n shWillin = 19; Day 8: n shScr = 18, n shScrRA = 12, n shWillin = 11). Groups were compared using Student’s t -test; ns: p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Membranes were blocked in either 5% skimmed dried milk in TBS-T or 5% BSA in TBS-T for 1 h at room temperature, before being incubated with primary antibody diluted in blocking solution overnight at 4°C [FRMD6: Cell Signaling Technology, 14688, 1:100; Phospho-p44/42 MAPK: Cell Signaling Technology, 4370, 1:4,000; p44/42 MAPK (ERK1/2): Cell Signaling Technology, 9107, 1:2,000; NeuroD1: Santa Cruz Biotechnology, sc-1084, 1:100; Vinculin: Novus Biologicals, NBP2-41274, 1:500; β-actin: Sigma–Aldrich, A1978, 1:20,000; GAPDH: Sigma–Aldrich, G8795, 1:20,000].

Techniques: Knockdown, Activation Assay, Western Blot, Control, Plasmid Preparation, Comparison, Imaging

Journal: Frontiers in Cellular Neuroscience

Article Title: Willin/FRMD6 Influences Mechanical Phenotype and Neuronal Differentiation in Mammalian Cells by Regulating ERK1/2 Activity

doi: 10.3389/fncel.2020.552213

Figure Lengend Snippet: Knock-down of Willin/FRMD6 alters vinculin expression, focal adhesion assembly, and actin cytoskeleton dynamics. (A) Representative phase-contrast images (upper row), immunostainings against F-actin (red), vinculin (green) and DNA (blue; middle row) and TAZ (lower row) of control ( shScr ) and Willin/FRMD6 knock-down ( shWillin ) SH-SY5Y cells. Scale bars: 30 μm. Comparison of (B) the number of focal adhesions per cell, (C) the mean area of a single focal adhesion, and (E) the nuclear/cytosolic TAZ ratio in shScr and shWillin cells. Each data point in (B) , (C) and (E) represents the measurement value for one cell taken from two independent experiments. Lines indicate means, error bars SEM. (D) Quantitative Western blot analysis of vinculin expression in shScr and shWillin cells. Means were calculated from three independent experiments. Error bars represent SEM. GAPDH serves as a loading control of the lysates. Correlation between the ratio of nuclear to cytoplasmic TAZ concentration, (F) the number of focal adhesions, and (G) the volume by which cells indent into the ERISM substrate for shScr (green dots) and shWillin (yellow dots) SH-SY5Y cells. Groups were compared using Student’s t -test; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Membranes were blocked in either 5% skimmed dried milk in TBS-T or 5% BSA in TBS-T for 1 h at room temperature, before being incubated with primary antibody diluted in blocking solution overnight at 4°C [FRMD6: Cell Signaling Technology, 14688, 1:100; Phospho-p44/42 MAPK: Cell Signaling Technology, 4370, 1:4,000; p44/42 MAPK (ERK1/2): Cell Signaling Technology, 9107, 1:2,000; NeuroD1: Santa Cruz Biotechnology, sc-1084, 1:100; Vinculin: Novus Biologicals, NBP2-41274, 1:500; β-actin: Sigma–Aldrich, A1978, 1:20,000; GAPDH: Sigma–Aldrich, G8795, 1:20,000].

Techniques: Knockdown, Expressing, Control, Comparison, Western Blot, Concentration Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Willin/FRMD6 Influences Mechanical Phenotype and Neuronal Differentiation in Mammalian Cells by Regulating ERK1/2 Activity

doi: 10.3389/fncel.2020.552213

Figure Lengend Snippet: Willin/FRMD6 over-expression results in a general reversal of the findings observed for knock-down cells and activate the mechanical activity of SH-SY5Y cells. (A) Quantitative Western blot analysis of Willin/FRMD6 for control ( Vector ) and Willin/FRMD6 overexpressing ( Willin ) SH-SY5Y cells. Means and SEM (error bars) were calculated from two independent experiments, each of which was conducted in triplicates. β-actin serves as a loading control of the lysates. (B) qPCR analysis of Willin/FRMD6 expression in Vector and Willin cells. Means and SEM (error bars) were calculated from two independent experiments, each of which was conducted in triplicates. (C) Growth curve of Vector and Willin cells. Means and SEM (error bars) were calculated from three independent experiments, each of which was conducted in triplicates. (D) Assessment of migration of shScr and shWillin cells in Boyden chambers after 24 h. Means (horizontal lines) and SEM (error bars) were calculated from two independent experiments, each of which was conducted in triplicates. (E) Representative phase-contrast images (upper row), immunostainings against F-actin (red), vinculin (green) and DNA (blue; middle row), and TAZ (lower row) of Vector and Willin SH-SY5Y cells. Scale bars: 30 μm. Comparison of (F) cell area, (G) cell elongation, (H) nuclear/cytosolic TAZ ratio, and (I) volume by which cells indent into the ERISM substrate for Vector and Willin cells. Each data point represents the value measured for one cell. Lines indicate means, error bars SEM. Groups were compared using Student’s t -test; ns: p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Membranes were blocked in either 5% skimmed dried milk in TBS-T or 5% BSA in TBS-T for 1 h at room temperature, before being incubated with primary antibody diluted in blocking solution overnight at 4°C [FRMD6: Cell Signaling Technology, 14688, 1:100; Phospho-p44/42 MAPK: Cell Signaling Technology, 4370, 1:4,000; p44/42 MAPK (ERK1/2): Cell Signaling Technology, 9107, 1:2,000; NeuroD1: Santa Cruz Biotechnology, sc-1084, 1:100; Vinculin: Novus Biologicals, NBP2-41274, 1:500; β-actin: Sigma–Aldrich, A1978, 1:20,000; GAPDH: Sigma–Aldrich, G8795, 1:20,000].

Techniques: Over Expression, Knockdown, Activity Assay, Western Blot, Control, Plasmid Preparation, Expressing, Migration, Comparison