Obinutuzumab (GA101) was generously supplied by Hoffmann La Roche (Switzerland). RTX was purchased from Genentech Inc. (South San Francisco, CA, USA). Antibodies for flow cytometry, CD20-PE (Clone HI47), CD20-FITC (Clone HI47) and CD19-FITC (SJ25-C1), were obtained from Invitrogen (CA, USA). Western blot antibodies, phospho-PLC gamma2 (Tyr759, 150 KDa), total PLC gamma2 (150 KDa), phospho GSK3 Beta (Ser21/9, 46 KDa), total GSK3 Beta (46KDa), phospho BTK (Tyr223, 78KDa), total BTK (Clone D3H5, 77KDa), phospho Lyn (Tr507, 53, 56KDa) and total Lyn (C13F9,58KDa), phospho B- RAF (Ser445, 86KDa), total B- RAF (clone 55C6, 86KDa), phospho ERK1/2 (Thr202/Thyr204) (Clone D1.14.4E, 42, 44KDa), total ERK1/2 (42,44KDa), phospho LCK (Tyr505, 56KDa), total LCK (56KDa) and B-actin (clone D6A8, 45KDa) were obtained from Cell Signaling Technology (Danvers, MA, USA). CD20 (B9E9) antibody was obtained by Santa Cruz Biotechnology (Dallas, TX, USA). Annexin V: PE Apoptosis Detection Kit I was obtained from BD Biosciences (San Jose, CA, USA). Alamar blue cell viability reagent was purchased from Invitrogen (Grand Island, NY, USA). U0126 MEK1/2 inhibitor was purchased from Cell Signaling Technology (Danvers, MA, USA). PLC gamma2 and GSK3 Beta ON-TARGET plus siRNA were obtained from Dharmacon GE (Lafayette, CO, USA).
Phospho b raf
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-B-Raf is a laboratory product that detects the phosphorylated form of the B-Raf protein. B-Raf is a serine/threonine-protein kinase that plays a role in the MAPK/ERK signaling pathway. The phosphorylation of B-Raf is an important regulatory mechanism for this protein.
Automatically generated - may contain errors
Lab products found in correlation
Total erk1 2, by Cell Signaling Technology (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
C raf, by Cell Signaling Technology (2 mentions)
Mek1 2, by Cell Signaling Technology (2 mentions)
Phospho lyn, by Cell Signaling Technology (1 mentions)
Phospho gsk 3 beta, by Cell Signaling Technology (1 mentions)
Phospho btk, by Cell Signaling Technology (1 mentions)
Total btk, by Cell Signaling Technology (1 mentions)
Total lck, by Cell Signaling Technology (1 mentions)
Mek1 2 inhibitor u0126, by Cell Signaling Technology (1 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Gadph, by Proteintech (1 mentions)
Ecl kit, by Advansta (1 mentions)
Il 25, by R&D Systems (1 mentions)
α smooth muscle actin α sma, by Proteintech (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease and phosphatase inhibitors, by Keygen Biotech (1 mentions)
5 protocols using phospho b raf
1
Obinutuzumab and Rituximab Signaling Analysis
2
Western Blot Analysis of Cell Signaling
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Protein lysates from cells receiving different treatments were prepared using RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (KeyGEN, Nanjing, China). Equal amounts of total protein (30 μg of protein per lane) were loaded onto gels for electrophoresis and then transferred onto PVDF membranes (Millipore, Billerica, USA), followed by incubation in blocking buffer (25 mM Tris, pH 7.4, 0.15 M NaCl, 0.1% Tween-20, 5% nonfat milk) for 1 h at RT. The membranes were incubated with primary antibodies against α-smooth muscle actin (α-SMA) (Proteintech, Chicago, USA), IL-25 (R&D, Minneapolis, USA), total ERK1/2, phospho-ERK1/2, total AKT, phospho-AKT, phospho-B-Raf (Cell Signaling Technology, Boston, USA) or GADPH (Proteintech, Chicago, USA) at 4 °C overnight. After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at RT. Proteins were visualized with an ECL kit (Advansta, CA, USA). GADPH was used as a loading control. The intensity of Western blotting bands was analyzed with Quantity One v4.6.2 software.
3
Investigating MAPK Pathway Signaling
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Antibody of EGF Receptor (#2232), Phospho-BRAF (Ser445, #2696), CRAF (#53745), Phospho-c-Raf (Ser338, #9427), MEK1/2 (#4694), MEK1 (#12671), MEK1 (#2352), MEK2 (#9147), p-MEK1/2 (Ser217/221, #9154), ERK1/2 (#4695), p-ERK1/2 (Thr202/Tyr204, #4370), GST-Tag (#2624), PD-L1 (#13684) were obtained from Cell Signaling Technology (Danvers, MA), BRAF V600E (#A1137-25) from Biovision (San Francisco, USA), and BRAF (#ab33899), Ki67 (#ab16667) obtained from Abcam (Cambridge, USA). β-Actin (#sc47778) provided by Santa Cruz Biotechnology (Santa Cruz, USA), and anti-Flag antibody (#F1804) was obtained from Sigma–Aldrich (St. Louis, USA). FITC Rat Anti-Mouse CD45 (#553079), PerCP-Cy™5.5-CD8a (#551162), APC-NK-1.1 (#550627), PE-CD4 (#557308) obtained from BD (New Jersey, USA), and in vivo anti-mouse PD-L1 (#BE0101) obtained from BioXcel, USA.
4
Immunoblotting Analysis of Signaling Pathways
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Ascites cells were lysed in buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 1 mM EDTA, 5 mM NaF and 5 mM Na3VO4. Solid tumor tissues were homogenized using the microtube pestles (Thomas Scientific) prior to add the lysis buffer above. Total cell lysates (30 µg) were resolved by 10% SDS–PAGE, transferred to nitrocellulose membranes, and incubated with 100–200 µg/mL of the following antibodies: phospho-Akt (Ser473), phospho-p44/42 ERK (Thr202/Tyr204), phospho-NF-κB p65 (Ser536), phospho-mTOR (Ser2448), phospho-B-Raf (Ser445), phospho-p38 MAPK (Thr180/Tyr182), phospho-Met (Tyr1234/1235) (Cell Signaling Technologies), KSHV-LANA (ABI) and K8.1A (Thermo Scientific-Pierce, Clone: 8C12G10G1). For loading controls, lysates were also incubated with antibodies detecting β-Actin (Sigma). Immunoreactive bands were developed using an enhanced chemiluminescence reaction (Perkin-Elmer).
5
Molecular Signaling Pathway Analysis
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MM cells were treated with or without novel or conventional agents, harvested, washed, and lysed as reported [32 (link),33 (link)]. Cell lysates were subjected to SDS-PAGE, transferred to membranes, and immunoblotted with the following antibodies: anti-p27, cyclin D1, phospho-B-Raf (Ser445), B-Raf, phospho-v-raf-1 murine leukemia viral oncogene homolog 1 (C-Raf) (Ser338), C-Raf, phospho-MEK1/2 (Ser217/221), MEK1/2, phospho-ERK (Thr202/Tyr204), ERK, phospho-v-akt murine thymoma viral oncogene homolog (Akt) (Ser473), Akt, caspase 3, poly (ADP-ribose) polymerase (PARP), β-actin, Bim, α-tubulin (all from Cell Signaling, Beverly, MA, USA), as well as NRAS and KRAS (both from Santa Cruz Biotechnology, Dallas, TX, USA).
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