rabbit polyclonal anti phosphorylated braf  (Cell Signaling Technology Inc)

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