Protocol detail

Western Blotting with Diverse Antibodies

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Western blotting was carried out as described previously 15 (link), 21 (link), 22 (link). Anti-Bcl-2, anti-Mcl-1, anti-PARP1, anti-cytochrome C, anti-ERK, anti-p-ERK, anti-Bim, anti-TSHR, anti-HK2, anti-Bax, anti-cyclin D1, and anti-GAPDH antibodies were purchased from ProteinTech (Chicago, IL, USA); anti-GLUT1 (NB110-39113SS) antibody was acquired from Novusbio. Anti-Beclin1 (#3738), anti-p62 (#8025), anti-p27 Kip1 (#3688), anti-Bcl-xl (#2764), anti-LC3A/B (#12741), anti-Caspase-3 (#9662), anti- Caspase-9 (#9502), anti-Caspase-8 (#9746) antibodies were purchased from Cell Signaling Technology. For immunoprecipitation, cells were treated for 24 hours with Vemurafenib before harvesting. NP40 lysates and BRAF immunoprecipitates were prepared according to the previous publication using rabbit anti-BRAF antibody (Santa Cruz Biotechnology, sc-5284) and analyzed by immunoblot using mouse anti-CRAF antibody (Santa Cruz Biotechnology, sc-227) or inversely 23 (link).

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Wei W.J., Sun Z.K., Shen C.T., Song H.J., Zhang X.Y., Qiu Z.L, & Luo Q.Y. (2017). Obatoclax and LY3009120 Efficiently Overcome Vemurafenib Resistance in Differentiated Thyroid Cancer. Theranostics, 7(4), 987-1001.

Publication 2017

Anti-Bcl-2 anti-Mcl-1 anti-PARP1 anti-cytochrome C anti-ERK anti-p-ERK anti-Bim anti-HK2 anti-Bax anti-cyclin D1 anti-GAPDH Anti-Beclin1 anti-p62 anti-p27 Kip1 anti-Bcl-xl anti-LC3A/B anti-Caspase-3 anti- Caspase-9 anti-Caspase-8

Anti antibodies Anti antibody Antibodies Antibody Bcl 2 Beclin1 Braf Caspase 3 Caspase 8 Caspase 9 Cells Craf Cyclin d1 Cytochrome c Gapdh Glut1 Immunoblot Immunoprecipitation Mouse P27 kip1 Parp1 Rabbit Vemurafenib

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Variable analysis

independent variables

Vemurafenib treatment duration (24 hours)

dependent variables

BRAF immunoprecipitation
CRAF binding to BRAF

control variables

NP40 lysates for immunoprecipitation
Rabbit anti-BRAF antibody for immunoprecipitation
Mouse anti-CRAF antibody for immunoblot

controls

Positive control: None mentioned.
Negative control: None mentioned.

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