Western Blot Analysis of Cell Signaling

Western blots were performed as previously described [54 (link)]. The following antibodies were used: IGFBP5 (ab4255, Abcam, Cambridge, UK), vimentin (ab8069, Abcam), GAPDH (ab75834, Abcam), HIF1α (NB100-134, Novus, St. Louis, MO, USA), IGF1R-P(Try1135/1136)(3024, Cell Signaling Technology (CST), Danvers, MA, USA), ERK1/2-P (Thr202/Tyr204) (4376, CST), ERK1/2 (4695, CST), E-cadherin (3195, CST), p38-MAPK-P (Thr180/Tyr182) (9215, CST), p38-MAPK (9212, CST), sheep anti-mouse IgG (ZB-2305, ZSGB-Bio, Beijing, China), and sheep anti-rabbit IgG (ZB-2301, ZSGB-Bio). Enhanced chemiluminescence substrate kit (RPN2232, GE Healthcare, Piscataway, NJ, USA) was used for the chemiluminescent detection of signals with BioMax film (Kodak, Rochester, NY, USA).

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Wang J., Ding N., Li Y., Cheng H., Wang D., Yang Q., Deng Y., Yang Y., Li Y., Ruan X., Xie F., Zhao H, & Fang X. (2015). Insulin-like growth factor binding protein 5 (IGFBP5) functions as a tumor suppressor in human melanoma cells. Oncotarget, 6(24), 20636-20649.

Publication 2015

IGFBP5 vimentin ab8069 GAPDH HIF1α ZB-2301 Enhanced chemiluminescence substrate kit BioMax film

Anti igg Antibodies Chemiluminescence E cadherin Erk1 Gapdh Hif1α Igf1r Igfbp5 Mouse Novus P38 mapk Rabbit Sheep Signals detection Vimentin Western blots

Corresponding Organization :

Other organizations : Beijing Institute of Genomics, Chinese Academy of Sciences, University of Chinese Academy of Sciences, People's Liberation Army No. 150 Hospital

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of IGFBP5, vimentin, GAPDH, HIF1α, IGF1R-P(Try1135/1136), ERK1/2-P (Thr202/Tyr204), ERK1/2, E-cadherin, p38-MAPK-P (Thr180/Tyr182), p38-MAPK

control variables

None explicitly mentioned

controls

Positive control: none mentioned
Negative control: none mentioned

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