Protocol detail

Western Blot Analysis of Cell Signaling Proteins

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Western blots were performed as described [52 (link)] using the following antibodies and dilutions: 1:1000 Uhrf1 (sc-373750, Santa Cruz Biotechnology), 1:1000 Rb (9313T, Cell Signaling), actin (A1978, Sigma), E2F1 (3742S, Cell Signaling), E2F2 (sc-9967, Santa Cruz Biotechnology), E2F3 (MA5-11319, Nalgene Nunc); 1:1000 SEMA3E (PA547469, Thermo Scientific); 1:1000 AMPK (5831S, Cell Signaling); 1:1000 pAMPK (ab133448, Abcam). Secondary antibodies were diluted 1:1000 (PI-1000, PI-2000 or PI-9500, Vector Laboratories). Bands were visualized using chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific). Band intensities were analyzed using ImageJ software.

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Wu S.C., Kim A., Gu Y., Martinez D.I., Zocchi L., Chen C.C., Lopez J., Salcido K., Singh S., Wu J., Nael A, & Benavente C.A. (2022). UHRF1 overexpression promotes osteosarcoma metastasis through altered exosome production and AMPK/SEMA3E suppression. Oncogenesis, 11(1), 51.

Publication 2022

AMPK (5831S, pAMPK PI-1000 PI-2000 PI-9500 SuperSignal West Pico Chemiluminescent Substrate

Actin Antibodies Chemiluminescence Dilutions E2f1 Sema3e Vector Western blots

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Variable analysis

independent variables

Antibody dilutions

dependent variables

Band intensities

control variables

Western blot procedure as described in reference [52]
Secondary antibody dilutions

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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