phosphorylated braf  (Cell Signaling Technology Inc)

Journal: Biomedicines

Article Title: Activation of MAP Kinase Pathway by Polyisoprenylated Cysteinyl Amide Inhibitors Causes Apoptosis and Disrupts Breast Cancer Cell Invasion

doi: 10.3390/biomedicines12030470

Figure Lengend Snippet: Proposed mechanism of action of the PCAIs in breast cancer cell lines. Abbreviations: RTK, Receptor Tyrosine Kinase; GRB2, Growth Factor Receptor Bound Protein 2; SOS, Son-of-Sevenless; GDP, Guanosine Diphosphate; GTP, Guanosine Triphosphate; RAS, Rat Sarcoma; BRAF, Rapidly Accelerated Fibrosarcoma, v-Raf Murine Sarcoma Viral Oncogene Homolog B; CRAF, RAF Proto-Oncogene Serine/Threonine-Protein Kinase; MEK, Mitogen-Activated Protein Kinase Kinase; ERK, Extracellular-Signal-Regulated Kinases; p90RSK, 90 kDa Ribosomal s6 Kinases; AKT, Protein Kinase B; BAK1, BCL2 Antagonist/Killer 1.

Article Snippet: Primary antibodies specific to phosphorylated proteins BRAF (p-BRAF, Cat #2696), CRAF (p-CRAF, Cat #9427), MEK 1/2 (p-MEK 1/2, Cat #9154S), ERK 1/2 (p-ERK1/2, Cat #4370S), p90RSK (p-p90RSK, Cat #11989S), AKT (p-AKT, Cat # 4060s), vinculin (E1E9V, Cat #13901), full-length Caspase 3 (Cat #9662S), full-length Caspase 7 (Cat #9492S), secondary antibodies anti-mouse IgG, HRP-linked antibody (Cat #7076), and anti-rabbit IgG, HRP-linked antibody (Cat #7074), and housekeeping protein GAPDH (Cat #5174S) or α-actinin (Cat #6478S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques:

Journal: International journal of molecular sciences

Article Title: Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations.

doi: 10.3390/ijms232214385

Figure Lengend Snippet: Figure 2. Detection of MKRN1-BRAF fusion by next-generation sequencing. (A) Representative part of NGS sequencing of the MKRN1–BRAF fusion in THJ-16T cells. Green part, BRAF gene; blue part, MKRN1 gene; red part, the MKRN1-BRAF fusion. (B) Schematics of wild-type BRAF (green), wild-type MKRN1 (blue), BRAFV600E (yellow), and the fused MKRN1-BRAF proteins. The zinc finger domains of MKRN1 and the serine-threonine (S/T) kinase domain of BRAF remain intact in the fused protein. WT, wild-type; ex, exon; RBD, Ras-binding domain; CRD, cysteine-rich domain.

Article Snippet: The membrane was blocked with 5% skimmed milk in tris-buffered saline (TBS-T) and incubated overnight at 4 ◦C with total rabbit polyclonal anti-ERK1/2 (1:500, WL01864, Wanleibio, Shenyang, China), rabbit polyclonal anti-phosphorylated ERK1/2 (1:200, WLP1512, Wanleibio, Shenyang, China), total mouse monoclonal anti-MEK1/2 (1:1000, sc-81504, Santa Cruz Biotech, CA, Canada), mouse monoclonal anti-phosphorylated MEK1/2 (1:1000, sc-81503, Santa Cruz Biotech, CA, Canada), rabbit monoclonal anti-N-term BRAF (1:1000, #14814, Cell Signaling, Topsfield, MA, USA), rabbit polyclonal anti-C-term BRAF (1:1000, OM160689, Omnimabs, Alhambra, CA, USA), rabbit polyclonal anti-phosphorylated BRAF (1:1000, #2696, Cell Signaling, MA, USA), rabbit polyclonal anti-phosphorylated STAT3 (1:500, WLP2412, Wanleibio, Shenyang, China), total rabbit polyclonal anti-STAT3 (1:300, WL03208, Wanleibio, Shenyang, China), rabbit polyclonal anti-IL-6 (1:1000, WL02841, Wanleibio, Shenyang, China), and rabbit polyclonal anti-GAPDH (1;5000, 10494-1-AP, Proteintech, Wuhan, China), followed by incubation with HRP-conjugated goat anti-rabbit IgG (SE134, Solarbio Life Sciences, Beijing, China) or HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, Wuhan, China).

Techniques: Next-Generation Sequencing, Sequencing, Binding Assay

Journal: International journal of molecular sciences

Article Title: Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations.

doi: 10.3390/ijms232214385

Figure Lengend Snippet: Figure 3. Identification of mutant BRAF expression by reverse transcription–polymerase chain reaction and Sanger sequencing. Reverse transcription–polymerase chain reaction (RT-PCR) detected BRAF exon 15 and MKRN1-BRAF transcripts in ATC cells. Sanger sequencing chromatograph of the BRAF exon 15 and MKRN1–BRAF fusion in THJ-11T (A), THJ-16T (B), THJ-21T (C), and Nthyori 3-1 cells (A,C). The arrow shows the breakpoint of the fusion between MKRN1 (NM_001145125, end of exon 3) and BRAF (NM_004333, start of exon 10).

Article Snippet: The membrane was blocked with 5% skimmed milk in tris-buffered saline (TBS-T) and incubated overnight at 4 ◦C with total rabbit polyclonal anti-ERK1/2 (1:500, WL01864, Wanleibio, Shenyang, China), rabbit polyclonal anti-phosphorylated ERK1/2 (1:200, WLP1512, Wanleibio, Shenyang, China), total mouse monoclonal anti-MEK1/2 (1:1000, sc-81504, Santa Cruz Biotech, CA, Canada), mouse monoclonal anti-phosphorylated MEK1/2 (1:1000, sc-81503, Santa Cruz Biotech, CA, Canada), rabbit monoclonal anti-N-term BRAF (1:1000, #14814, Cell Signaling, Topsfield, MA, USA), rabbit polyclonal anti-C-term BRAF (1:1000, OM160689, Omnimabs, Alhambra, CA, USA), rabbit polyclonal anti-phosphorylated BRAF (1:1000, #2696, Cell Signaling, MA, USA), rabbit polyclonal anti-phosphorylated STAT3 (1:500, WLP2412, Wanleibio, Shenyang, China), total rabbit polyclonal anti-STAT3 (1:300, WL03208, Wanleibio, Shenyang, China), rabbit polyclonal anti-IL-6 (1:1000, WL02841, Wanleibio, Shenyang, China), and rabbit polyclonal anti-GAPDH (1;5000, 10494-1-AP, Proteintech, Wuhan, China), followed by incubation with HRP-conjugated goat anti-rabbit IgG (SE134, Solarbio Life Sciences, Beijing, China) or HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, Wuhan, China).

Techniques: Mutagenesis, Expressing, Reverse Transcription, Polymerase Chain Reaction, Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: International journal of molecular sciences

Article Title: Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations.

doi: 10.3390/ijms232214385

Figure Lengend Snippet: Figure 4. Differential expression of pBRAF, BRAF, pMEK, MEK, pERK, ERK in Nthyori 3-1, THJ-11T, THJ-16T, and THJ-21T cells. (A) Western blotting analyses of pBRAF, BRAF, pMEK, MEK, pERK, and ERK levels in Nthyori 3-1, THJ-16T, and THJ-21T cells. GAPDH served as a loading control. (B) Immunocytochemical staining (scale bar, 5 µm) of pERK performed on Nthyori 3-1, THJ-11T, THJ-16T, and THJ-21T cells.

Article Snippet: The membrane was blocked with 5% skimmed milk in tris-buffered saline (TBS-T) and incubated overnight at 4 ◦C with total rabbit polyclonal anti-ERK1/2 (1:500, WL01864, Wanleibio, Shenyang, China), rabbit polyclonal anti-phosphorylated ERK1/2 (1:200, WLP1512, Wanleibio, Shenyang, China), total mouse monoclonal anti-MEK1/2 (1:1000, sc-81504, Santa Cruz Biotech, CA, Canada), mouse monoclonal anti-phosphorylated MEK1/2 (1:1000, sc-81503, Santa Cruz Biotech, CA, Canada), rabbit monoclonal anti-N-term BRAF (1:1000, #14814, Cell Signaling, Topsfield, MA, USA), rabbit polyclonal anti-C-term BRAF (1:1000, OM160689, Omnimabs, Alhambra, CA, USA), rabbit polyclonal anti-phosphorylated BRAF (1:1000, #2696, Cell Signaling, MA, USA), rabbit polyclonal anti-phosphorylated STAT3 (1:500, WLP2412, Wanleibio, Shenyang, China), total rabbit polyclonal anti-STAT3 (1:300, WL03208, Wanleibio, Shenyang, China), rabbit polyclonal anti-IL-6 (1:1000, WL02841, Wanleibio, Shenyang, China), and rabbit polyclonal anti-GAPDH (1;5000, 10494-1-AP, Proteintech, Wuhan, China), followed by incubation with HRP-conjugated goat anti-rabbit IgG (SE134, Solarbio Life Sciences, Beijing, China) or HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, Wuhan, China).

Techniques: Quantitative Proteomics, Western Blot, Control, Staining

Journal: International journal of molecular sciences

Article Title: Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations.

doi: 10.3390/ijms232214385

Figure Lengend Snippet: Figure 5. Differential pBRAF, BRAF, pMEK, MEK, pERK, ERK expression in THJ-11T, THJ-16T, and THJ-21T cells without and with resveratrol (R), trametinib (T), dabrafenib and trametinib (D+T), dabrafenib (D) and/or trametinib (T) in combination with resveratrol (R) treatment. (A) Western blotting was performed on the sample proteins of THJ-11T, THJ-16T, and THJ-21T cells before and after 48h drug treatment, GAPDH served as a loading control. (B) pERK immunocytochemical staining (scale bar, 5 µm) performed on THJ-11T, THJ-16T, and THJ-21T cells after 48h drugs treatment.

Article Snippet: The membrane was blocked with 5% skimmed milk in tris-buffered saline (TBS-T) and incubated overnight at 4 ◦C with total rabbit polyclonal anti-ERK1/2 (1:500, WL01864, Wanleibio, Shenyang, China), rabbit polyclonal anti-phosphorylated ERK1/2 (1:200, WLP1512, Wanleibio, Shenyang, China), total mouse monoclonal anti-MEK1/2 (1:1000, sc-81504, Santa Cruz Biotech, CA, Canada), mouse monoclonal anti-phosphorylated MEK1/2 (1:1000, sc-81503, Santa Cruz Biotech, CA, Canada), rabbit monoclonal anti-N-term BRAF (1:1000, #14814, Cell Signaling, Topsfield, MA, USA), rabbit polyclonal anti-C-term BRAF (1:1000, OM160689, Omnimabs, Alhambra, CA, USA), rabbit polyclonal anti-phosphorylated BRAF (1:1000, #2696, Cell Signaling, MA, USA), rabbit polyclonal anti-phosphorylated STAT3 (1:500, WLP2412, Wanleibio, Shenyang, China), total rabbit polyclonal anti-STAT3 (1:300, WL03208, Wanleibio, Shenyang, China), rabbit polyclonal anti-IL-6 (1:1000, WL02841, Wanleibio, Shenyang, China), and rabbit polyclonal anti-GAPDH (1;5000, 10494-1-AP, Proteintech, Wuhan, China), followed by incubation with HRP-conjugated goat anti-rabbit IgG (SE134, Solarbio Life Sciences, Beijing, China) or HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, Wuhan, China).

Techniques: Expressing, Western Blot, Control, Staining

Journal: Nature

Article Title: Structure of the MRAS–SHOC2–PP1C phosphatase complex

doi: 10.1038/s41586-022-05086-1

Figure Lengend Snippet: a , SPR-derived affinity and cooperativity immobilizing NRAS–GppNHp, NRAS(Q61R)–GTP, KRAS–GppNHp or KRAS(Q61R)–GTP with the indicated analytes. α = K D SHOC2 / K D SHOC2–PP1C . Affinity values over 10,000 nM are highlighted in red. b , In vitro dephosphorylation of autoinhibited and active-state BRAF–MEK1–14-3-3 complexes by the SHOC2 holophosphatase. Purified full-length BRAF complexes in the autoinhibited state (left) or active dimeric state (right) were incubated with lambda phosphatase (PPase), PP1Cα or ternary SHOC2 complexes, and were blotted with phospho-specific antibodies for BRAF phosphorylated at Ser365 and Ser729. Equivalent loading of BRAF complexes is shown by Coomassie staining. Phosphorylated Ser365 is selectively dephosphorylated relative to Ser729 in the active dimer, whereas both are relatively protected in the autoinhibited (14-3-3-bound) state. Experiments were conducted twice with similar results. c , The relationship between the knockdown of SHOC2 and MRAS , HRAS , NRAS and KRAS . The dependency scores of each RAS gene and SHOC2 are shown on the x and y axes, respectively. Dashed lines indicate a dependency score of zero. A highly negative dependency score implies that a given cell line is highly dependent on that gene. Cell lines dependent on both SHOC2 and RAS are indicated at the bottom left. Right, the calculated Pearson correlation coefficient ( y axis) applied to each mutation group (group of cell lines containing the associated mutation). A higher positive value indicates a stronger positive relationship: the dependency score of SHOC2 decreases/increases in the same lines as the dependency scores of the RAS genes. The n values above each bar show the number of cell lines in each mutation group. d , Immunoblot analysis of MiaPaca2 parental cells (Par), SHOC2- KO (KO) and stable cell lines reconstituted with SHOC2 mutants after 10 nM trametinib (Tram) treatment for 1 h or 24 h (+). Densitometry quantification (percentage variation) of pCRAF/CRAF and pERK/ERK levels from the immunoblot analysis normalized to untreated MiaPaca2 parental cells. The samples were derived from the same experiment and blots were processed in parallel. The images are representative of two independent experiments.

Article Snippet: Reaction products were analysed using western blotting using phosphospecific antibodies for the BRAF phosphorylated at Ser365 (Cell Signaling Technology, 9421S) or Ser729 (Abcam, ab124794).

Techniques: Derivative Assay, In Vitro, De-Phosphorylation Assay, Purification, Incubation, Staining, Mutagenesis, Western Blot, Stable Transfection