Western Blot Analysis of ERK1/2 Activation

ERK1/2 activation was evaluated using Western blotting. The procedure was carried out in the same manner as described in our earlier work [52 (link)]. Briefly, cultured cells in the 6-well plates were lysed in RIPA buffer (Sigma, Ronkonkoma, NY, USA) containing cocktails of protease and phosphatase inhibitors (Sigma, Ronkonkoma, NY, USA). The protein concentration was measured using DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Luminescence was detected by means of ChemiDoc XRS+ system (Bio-Rad, Hercules, CA, USA), and the luminescence intensity was calculated with Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA).

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Guryleva M.V., Chistyakov D.V., Lopachev A.V., Goriainov S.V., Astakhova A.A., Timoshina Y.A., Khutorova A.V., Fedorova T.N, & Sergeeva M.G. (2021). Modulation of the Primary Astrocyte-Enriched Cultures’ Oxylipin Profiles Reduces Neurotoxicity. Metabolites, 11(8), 498.

Publication 2021

RIPA buffer cocktails of protease and phosphatase inhibitors DC Protein Assay Kit ChemiDoc XRS+ system Image Lab 3.0 software

Assay Buffer Cultured cells Erk1 Inhibitors Luminescence Phosphatase Protease Protein Ripa

Corresponding Organization : Lomonosov Moscow State University

Other organizations : Research Center of Neurology, Peoples' Friendship University of Russia

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

ERK1/2 activation

control variables

Cell culture conditions (6-well plates)
Lysis buffer (RIPA buffer with protease and phosphatase inhibitors)
Protein concentration measurement (DC Protein Assay Kit)
Luminescence detection (ChemiDoc XRS+ system)
Luminescence intensity calculation (Image Lab 3.0 software)

controls

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

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