mdm2 d 7  (Santa Cruz Biotechnology)

Journal: Communications Biology

Article Title: Tau affects P53 function and cell fate during the DNA damage response

doi: 10.1038/s42003-020-0975-4

Figure Lengend Snippet: a , b Parental (wt) or 232P (Tau-KO) cells treated 30 min without (basal) or with 60 μM etoposide and recovered for 6 h in the absence (eto) or presence of 10 µg/mL KU-55933 and/or 5 µg/mL nutlin-3. a Mean intensity ± sem of single-cell nuclear P53 or MDM2 (DAPI mask, ImageJ) shown as fold of basal conditions, n > 100 cells/condition distributed over five images. b Percent clCasp3-positive cells shown as mean ± SD of five images (basal) or 15 images (treatments), n > 500 cells/condition. Non-parametric independent samples test and Kruskal–Wallis pairwise comparison between cell lines (in bold) or for treatment for each cell line (in vertical). c Western bot analysis of P53 in parental (wt) or 232 P (Tau-KO) cells at basal conditions, after 30 min 60 μM etoposide and 4 h recovery without or with 10 μM MG132. GAPDH served as loading control. d Parental (wt) or 232P (Tau-KO) cells pre-treated for 30 min with 60 μM etoposide followed by 4 h with 10 μM MG132, were incubated with 25 μM of cycloheximide (CHX) for the indicated chase times. Single-cell nuclear P53 or nuclear MDM2 (DAPI mask, ImageJ) shown as fold of wt cells at basal conditions. Mean intensity ± sem of n > 100 cells/condition distributed over five images. Independent measures ordinary two-way ANOVA, source of variation for cell lines (bold), multiple Bonferroni pairwise comparisons of treatment for each line (in italic). e Parental (wt) or (Tau-KO) 232P cells treated for 30 min with 60 μM etoposide and 6 h recovery analyzed with a 90 kDa MDM2 rabbit antibody (green and middle panel with GAPDH as loading control) or a 60 and 90 kDa MDM2 mouse antibody (red and bottom panel).

Article Snippet: The cell lysates for P53 immune precipitation were cleared by centrifugation at 20,000 g per 10 min. For immune blots , we used 0.2 μg/mL Tau13 (sc-21796, Santa Cruz), 0.18 μg/mL GAPDH (ab181602, Abcam), 0.4 μg/mL P53 DO-1 (sc-126, Santa Cruz), 4 μg/mL P53 Pab 1801 (sc-98, Santa Cruz), 0.5 μg/mL pS 15 -P53 (ab223868, Abcam), 0.1 μg/mL rabbit D1V2Z MDM2 (#86934, Cell Signaling), 0.2 μg/mL mouse SMP14 MDM2 (sc965, Santa Cruz), 0.05 μg/mL clAsp 175 -Caspase3 (#9661, Cell Signaling), or 0.2 μg/mL p21 (sc53870, Santa Cruz).

Techniques: Comparison, Western Blot, Control, Incubation

Journal: Communications Biology

Article Title: Tau affects P53 function and cell fate during the DNA damage response

doi: 10.1038/s42003-020-0975-4

Figure Lengend Snippet: Cell lysates of SH-SY5Y treated with 10 μM of MG132 to stabilize P53 expression, without (ctrl) or with (eto) a 30 min pre-treatment with 60 μM etoposide, were subjected to immune precipitation of endogenous P53 with a rabbit antibody (P53) or with a rabbit GFP antibody as negative IP control (GFP). Western blot analysis for co-precipitation of MDM2 or Tau with the respective mouse antibodies as indicated. The blots on the top show the analysis of the starting material (cell lysates), those on the bottom the immunoprecipitation (IP). The P53 blots are entirely shown, whereas for MDM2 and Tau, the blots were cut between the 55 kDa and the 95 kDa protein size markers and analyzed separately.

Article Snippet: The cell lysates for P53 immune precipitation were cleared by centrifugation at 20,000 g per 10 min. For immune blots , we used 0.2 μg/mL Tau13 (sc-21796, Santa Cruz), 0.18 μg/mL GAPDH (ab181602, Abcam), 0.4 μg/mL P53 DO-1 (sc-126, Santa Cruz), 4 μg/mL P53 Pab 1801 (sc-98, Santa Cruz), 0.5 μg/mL pS 15 -P53 (ab223868, Abcam), 0.1 μg/mL rabbit D1V2Z MDM2 (#86934, Cell Signaling), 0.2 μg/mL mouse SMP14 MDM2 (sc965, Santa Cruz), 0.05 μg/mL clAsp 175 -Caspase3 (#9661, Cell Signaling), or 0.2 μg/mL p21 (sc53870, Santa Cruz).

Techniques: Expressing, Control, Western Blot, Immunoprecipitation

Journal: Communications Biology

Article Title: Tau affects P53 function and cell fate during the DNA damage response

doi: 10.1038/s42003-020-0975-4

Figure Lengend Snippet: PCR Primers (all specific for homo sapiens mRNAs).

Article Snippet: The cell lysates for P53 immune precipitation were cleared by centrifugation at 20,000 g per 10 min. For immune blots , we used 0.2 μg/mL Tau13 (sc-21796, Santa Cruz), 0.18 μg/mL GAPDH (ab181602, Abcam), 0.4 μg/mL P53 DO-1 (sc-126, Santa Cruz), 4 μg/mL P53 Pab 1801 (sc-98, Santa Cruz), 0.5 μg/mL pS 15 -P53 (ab223868, Abcam), 0.1 μg/mL rabbit D1V2Z MDM2 (#86934, Cell Signaling), 0.2 μg/mL mouse SMP14 MDM2 (sc965, Santa Cruz), 0.05 μg/mL clAsp 175 -Caspase3 (#9661, Cell Signaling), or 0.2 μg/mL p21 (sc53870, Santa Cruz).

Techniques: