Western Blot Analysis of ALDH1L1 in NIH3T3 Cells

NIH3T3 cells were lysed in RIPA buffer containing 1x protease inhibitor cocktail (Sigma). Cell lysates were subjected to SDS-PAGE (20 μg of the total protein loaded per lane) followed by Western blot assays with corresponding antibodies. ALDH1L1 was detected using in-house rabbit polyclonal antibody (1:10000) generated against purified recombinant rat ALDH1L1 [7 (link), 16 (link)]. Primary antibodies used in this study are listed in S1 Table. All in-house antibodies have been verified in our previous studies [5 (link), 17 (link), 18 (link)]. HRP-conjugated secondary goat anti-rabbit IgG antibody and goat anti-mouse IgG antibody were from Abcam (both used at 1:7000). A Hybond TM-ECL nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburg, PA, USA) and Pierce ECL detection kit (ThermoFisher Scientific, Waltham, MA, USA) were used.

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Khan Q.A., Pediaditakis P., Malakhau Y., Esmaeilniakooshkghazi A., Ashkavand Z., Sereda V., Krupenko N.I, & Krupenko S.A. (2018). CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase. PLoS ONE, 13(7), e0199699.

Publication 2018

protease inhibitor cocktail goat anti-mouse IgG antibody Hybond TM-ECL nitrocellulose membrane Pierce ECL detection kit

Anti igg Antibodies Antibody Buffer Cell Goat House rabbit Membrane Mouse Nih3t3 cells Nitrocellulose Protease inhibitor Protein Rabbit Ripa Sds page Western blot assays

Corresponding Organization : University of North Carolina at Chapel Hill

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Variable analysis

independent variables

Presence of RIPA buffer containing 1x protease inhibitor cocktail (Sigma)

dependent variables

Expression of ALDH1L1 protein detected by Western blot

control variables

Amount of total protein loaded per lane (20 μg)
Primary antibodies used (listed in S1 Table)
Secondary antibodies used (HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG from Abcam, both used at 1:7000)
Nitrocellulose membrane (Hybond TM-ECL) and detection kit (Pierce ECL) used

controls

Positive control: In-house rabbit polyclonal antibody (1:10000) generated against purified recombinant rat ALDH1L1
Negative controls: Not explicitly mentioned

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