9421 ser338 phospho craf ser338  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Actin-modulated nuclear shape controls adaptive reprogramming in cancer-associated fibroblasts

doi: 10.1101/2025.07.19.665630

Figure Lengend Snippet: (A) Western blot confirming effective silencing of ARAF, BRAF, or CRAF expression in iM27 cells using corresponding siRNAs. (B) Representative fluorescence microscopy images showing β-catenin expression in iM27 cells transfected with scramble siRNA or siRNAs that deplete ARAF, BRAF, or CRAF expression under PLX4032 treatment. iM27 transfected with scramble siRNA without PLX4032 treatment were used as a control. (C) Quantification of nuclear β-catenin intensity in iM27 and genetically modified iM27 cells as shown in (B) with or without PLX4032 treatment using ImageJ. The data are presented as the means ± SDs (n = 11 randomly selected 20 X fields per group). (D) Representative PLA images showing BRAF-CRAF heterodimerization in DMSO- and PLX4032-treated iM27 cells. Red dots indicate BRAF-CRAF dimers. (E) Quantification of PLA signals comparing PLX4032-treated iM27 cells with DMSO-treated iM27 cells. The data are presented as the means ± SDs (n = 9-10 randomly selected 40X fields per group). (F) Representative PLA images showing BRAF-BRAF homodimerization in DMSO- and PLX4032-treated iM27 cells. The red dots indicate BRAF-BRAF dimers. (G) Quantification of PLA signals comparing PLX4032-treated iM27 cells with DMSO-treated iM27 cells. The data are presented as the means ± SDs (n = 9-10 randomly selected 40X fields per group). (H) Representative PLA images showing CRAF-CRAF homodimerization in DMSO- and PLX4032-treated iM27 cells. The red dots indicate CRAF-CRAF dimers. (I) Quantification of PLA signals comparing PLX4032-treated iM27 cells with DMSO-treated iM27 cells. The data are presented as the means ± SDs (n = 9-10 randomly selected 40X fields per group). (J) Western blot analysis showing increased CRAF-CRAF and CRAF-BRAF interactions in PLX4032-treated iM27 cells compared with those in DMSO-treated iM27 cells. Co-IP was performed via the use of an anti-Myc antibody to pull down interacting proteins in iM27 cells coexpressing Myc-tagged and Flag-tagged CRAF. (K-M) Western blot analysis of phosphorylated BRAF (Ser445) and CRAF (Ser338) expression in iM27 (K), BRAF-deficient iM27 overexpressing wild-type BRAF or BRAF (T529N) (L), and CRAF-deficient iM27 overexpressing wild-type CRAF or CRAF (T421N) (M) treated with either DMSO or PLX4032.

Article Snippet: The membranes were blocked with 5% fat-free milk or 5% BSA in TBST for one hour at room temperature and then incubated with the following primary antibodies: ARAF (Cell Signaling Technology, 4432, 1:1000), BRAF (Proteintech, 20899-1-AP, 1:1000), CRAF (Proteintech, clone 1D6A1, 1:1000), CRAF (Proteintech, 26863-1-AP, 1:1000), phospho-BRAF (Cell Signaling Technology, 2696, 1:1000), phospho-CRAF (Cell Signaling Technology, 9127, 1:1000), Flag (Proteintech, 20543-1-AP, 1:1000), MYC (Proteintech, clone 1A5A2, 1:1000), MYC (Cell Signaling Technology, clone 71D10, 1:1000), KRAS (Proteintech, 12063-1-AP, 1:1000), HRAS (Proteintech, 18295-1-AP, 1:1000), NRAS (Proteintech, 10724-1-AP, 1:1000), ERK1/2 (Cell Signaling Technology, clone 3A7, 1:1000), and phospho-ERK (Cell Signaling Technology, clone D13.14) in blocking buffer.

Techniques: Western Blot, Expressing, Fluorescence, Microscopy, Transfection, Control, Genetically Modified, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Actin-modulated nuclear shape controls adaptive reprogramming in cancer-associated fibroblasts

doi: 10.1101/2025.07.19.665630

Figure Lengend Snippet: (A) Western blot confirming effective silencing of KRAS, HRAS, and NRAS expression in iM27 using siRNAs. (B) PLA results showing BRAF and CRAF heterodimerization in iM27 transfected with scramble siRNA (Scr), scramble iM27 treaded with PLX4032, and RAS-deficient iM27 (siRASs) treated with PLX4032. (C) Western blot showing the phosphorylation of BRAF (Ser445) and CRAF (Ser338) in iM27 transfected with scramble siRNA and RAS-deficient iM27 with and without PLX4032 treatment. (D) Confocal images of nuclei visualized by Hoechst staining in iM27 transfected with scramble siRNA, scramble iM27 treaded with PLX4032, and RAS-deficient iM27 (siRASs) treated with PLX4032. The nuclear boundaries are outlined with yellow circles. (E-F) Quantification of nuclear morphology based on the confocal images shown in (D), including the nuclear aspect ratio (E) and circularity (F), was performed via ImageJ. The data are presented as the means ± SDs (n = 30-67 nuclei per group). (G) Confocal images of F-actin expression and organization in iM27 transfected with scramble siRNA, scramble iM27 treaded with PLX4032 (scramble), and RAS-deficient iM27 (siRASs) treated with PLX4032. Insets display enlarged views of representative individual cells (highlighted by yellow boxes). Yellow arrows indicate actin caps. (H-I) Quantification of F-actin intensity (H) and actin cap intensity (I) using ImageJ. The data are presented as the means ± SDs (n = 6 randomly selected 20 X fields per group for H; n = 25-52 nuclei per group for I). (J) Confocal images showing Nesprin-2 distribution in iM27 transfected with scramble siRNA, scramble iM27 treaded with PLX4032, and RAS-deficient iM27 (siRASs) treated with PLX4032. The yellow arrow indicates abnormal cytosolic localization of Nesprin-2. (K) Representative fluorescence images showing nuclear β-catenin staining in iM27 transfected with scramble siRNA, scramble iM27 treaded with PLX4032, and RAS-deficient iM27 (siRASs) treated with PLX4032. (L) Quantification of nuclear β-catenin intensity under the indicated conditions shown in (K). The data are presented as the means ± SDs (n = 9 randomly selected 20X fields per group).

Article Snippet: The membranes were blocked with 5% fat-free milk or 5% BSA in TBST for one hour at room temperature and then incubated with the following primary antibodies: ARAF (Cell Signaling Technology, 4432, 1:1000), BRAF (Proteintech, 20899-1-AP, 1:1000), CRAF (Proteintech, clone 1D6A1, 1:1000), CRAF (Proteintech, 26863-1-AP, 1:1000), phospho-BRAF (Cell Signaling Technology, 2696, 1:1000), phospho-CRAF (Cell Signaling Technology, 9127, 1:1000), Flag (Proteintech, 20543-1-AP, 1:1000), MYC (Proteintech, clone 1A5A2, 1:1000), MYC (Cell Signaling Technology, clone 71D10, 1:1000), KRAS (Proteintech, 12063-1-AP, 1:1000), HRAS (Proteintech, 18295-1-AP, 1:1000), NRAS (Proteintech, 10724-1-AP, 1:1000), ERK1/2 (Cell Signaling Technology, clone 3A7, 1:1000), and phospho-ERK (Cell Signaling Technology, clone D13.14) in blocking buffer.

Techniques: Western Blot, Expressing, Transfection, Phospho-proteomics, Staining, Fluorescence

Journal: bioRxiv

Article Title: Actin-modulated nuclear shape controls adaptive reprogramming in cancer-associated fibroblasts

doi: 10.1101/2025.07.19.665630

Figure Lengend Snippet: (A) Western blot showing increased ERK phosphorylation in response to increasing concentrations of PLX4032 in iM27 cells. (B) Western blot showing the phosphorylation of ERK in iM27 transfected with scramble siRNA and RAS-deficient iM27 (siRASs) with and without PLX4032 treatment. (C-D) Western blots showing ERK phosphorylation in BRAF-deficient iM27 overexpressing wild-type BRAF and BRAF(T529N) (C) and in CRAF-deficient iM27 overexpressing wild-type CRAF and CRAF(T421N) (D) with and without PLX4032 treatment. (E) Western blot showing increased phosphorylation of MYPT1 in PLX4032-treated iM27. (F) Western blot showing the phosphorylation of MYPT1 in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ERK inhibitor SCH772984 (SCH). (G) Representative confocal images of nuclear morphology visualized by Hoechst staining in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ROCK inhibitor Y27632. (H-I) Quantification of nuclear morphology based on the confocal images shown in (G), including the nuclear aspect ratio (H) and circularity (I), was performed via ImageJ. The data are presented as the means ± SDs (n = 27-38 nuclei per group). (J) Confocal images of F-actin expression and organization in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ROCK inhibitor Y27632. Insets display enlarged views of representative individual cells (highlighted by yellow boxes). Yellow arrows indicate the actin caps. (K) Quantification of the actin cap intensity via ImageJ shown in (J). The data are presented as the means ± SDs (n = 20 nuclei per group). (L) Confocal images showing SUN2 distribution in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ROCK inhibitor Y27632. The yellow arrow indicates abnormal nuclear localization of SUN2 in PLX4032-treated cells. (M) Representative fluorescence images showing nuclear β-catenin staining in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ROCK inhibitor Y27632. (N) Quantification of the nuclear β-catenin intensity under the indicated conditions shown in (M). The data are presented as the means ± SDs (n = 40 nuclei per group).

Article Snippet: The membranes were blocked with 5% fat-free milk or 5% BSA in TBST for one hour at room temperature and then incubated with the following primary antibodies: ARAF (Cell Signaling Technology, 4432, 1:1000), BRAF (Proteintech, 20899-1-AP, 1:1000), CRAF (Proteintech, clone 1D6A1, 1:1000), CRAF (Proteintech, 26863-1-AP, 1:1000), phospho-BRAF (Cell Signaling Technology, 2696, 1:1000), phospho-CRAF (Cell Signaling Technology, 9127, 1:1000), Flag (Proteintech, 20543-1-AP, 1:1000), MYC (Proteintech, clone 1A5A2, 1:1000), MYC (Cell Signaling Technology, clone 71D10, 1:1000), KRAS (Proteintech, 12063-1-AP, 1:1000), HRAS (Proteintech, 18295-1-AP, 1:1000), NRAS (Proteintech, 10724-1-AP, 1:1000), ERK1/2 (Cell Signaling Technology, clone 3A7, 1:1000), and phospho-ERK (Cell Signaling Technology, clone D13.14) in blocking buffer.

Techniques: Western Blot, Phospho-proteomics, Transfection, Staining, Expressing, Fluorescence