prlr  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Targeting of PTP4A3 overexpression sensitises HGSOC cells towards chemotherapeutic drugs

doi: 10.1101/2024.10.28.620719

Figure Lengend Snippet: ( A ) Expression of PTP4A3 was assessed in OVCAR 3, OVCAR 4 and Kuramochi cells by Western immunoblot and ( B ) this was quantified by densitometry, with PTP4A3 normalised to β-actin. ( C ) NCBI GEO repository RNA-seq data was analysed for gene expression in the three HGSOC cell lines, normalised to Kuramochi levels. ( D ) Cell lysates were analysed for phosphorylation of AKT/PKB (pSer473) and ERK1-2 (pThr202- Tyr204). Antibodies that recognise total AKT/PKB and ERK1-2 were used as controls. Expression of PTP4A3 is shown for comparison. ( E ) Turnover of PTP4A3 was analysed by incubating with or without either MG132 (2.5 µM, 2 h) or BafA1 (100 nM, 1 h) before lysates were collected with β-actin as loading control. Results are representative of at least three independent experiments. Error bars = ± S.E.M. **** p< 0.0001 by unpaired t -test.

Article Snippet: Membranes were incubated overnight at 4 °C on a rocker with the following antibodies in Blocking buffer: β-actin (Sigma-Aldrich, Gillingham, UK A5441), LC3B (Cell Signaling Technology, Leiden, Netherlands 2775), S6K1 (9202), p-S6K1 (9205), ERK1/2 (9102), p-ERK1/2 (9106), ULK1 (4773), p-ULK1 (6888), p-AKT (9271), PTP4A3 (Santa Cruz Biotechnology, Heidelberg, Germany sc-130355), AKT , pan-PTP4A3 (Bio-Techne MAB32191), and GFP (Thermo Fisher Scientific, Horsham, UK A-11122), then washed thrice with TBS-T and incubated with species-appropriate HRP- conjugated secondary antibodies (Thermo Fisher Scientific) diluted in Blocking buffer.

Techniques: Expressing, Western Blot, RNA Sequencing, Gene Expression, Phospho-proteomics, Comparison, Control

Journal: bioRxiv

Article Title: Targeting of PTP4A3 overexpression sensitises HGSOC cells towards chemotherapeutic drugs

doi: 10.1101/2024.10.28.620719

Figure Lengend Snippet: OVCAR 3 cells were transiently transfected with either pEGFP (empty vector), pEGFP-PTP4A1 or pEGFP-PTP4A3. ( A ) Cells were incubated with or without 100 nM BafA1 for 1 h. The phosphorylation of S6K1 (pThr389) was used to confirm the inhibition of mTORC1 signalling and β- actin was used as a loading control, while GFP was probed as a transfection efficiency control. ( B ) Basal autophagy activity was quantified by densitometry of LC3B-II and normalised to β-actin. ( C ) Alternatively, cells were incubated with or without EBSS for 2 h in the presence or absence of BafA1 (100 nM) for the last hour. ( D ) Activatable autophagy was quantified by densitometry of LC3B-II normalised to β-actin expression. Data was pooled from three independent experiments and error bars = ±S.E.M. * p< 0.05, *** p< 0.001, **** p< 0.0001 by two-way ANOVA test.

Article Snippet: Membranes were incubated overnight at 4 °C on a rocker with the following antibodies in Blocking buffer: β-actin (Sigma-Aldrich, Gillingham, UK A5441), LC3B (Cell Signaling Technology, Leiden, Netherlands 2775), S6K1 (9202), p-S6K1 (9205), ERK1/2 (9102), p-ERK1/2 (9106), ULK1 (4773), p-ULK1 (6888), p-AKT (9271), PTP4A3 (Santa Cruz Biotechnology, Heidelberg, Germany sc-130355), AKT , pan-PTP4A3 (Bio-Techne MAB32191), and GFP (Thermo Fisher Scientific, Horsham, UK A-11122), then washed thrice with TBS-T and incubated with species-appropriate HRP- conjugated secondary antibodies (Thermo Fisher Scientific) diluted in Blocking buffer.

Techniques: Transfection, Plasmid Preparation, Incubation, Phospho-proteomics, Inhibition, Control, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Targeting of PTP4A3 overexpression sensitises HGSOC cells towards chemotherapeutic drugs

doi: 10.1101/2024.10.28.620719

Figure Lengend Snippet: Kuramochi ( A ) and OVCAR 4 ( B ) cells were infected with lentiviral particles harbouring either scrambled control shRNA (Scr) or PTP4A3-targeting shRNA (shPTP4A3). The silencing of PTP4A3 expression was quantified by Western immunoblotting. ( C, D ) Kuramochi-Scr (K- Scr), ( E, F ) Kuramochi-shPTP4A3 (K-shPTP4A3), ( G, H ) OVCAR 4-Scr (4-Scr) and ( I, J ) OVCAR 4-shPTP4A3 (4-shPTP4A3) cells were incubated with EBSS for 0, 1, 2 or 4 h and BafA1 (100 nM) was added for the final hour. ( C, E, G, I ) The phosphorylation of S6K1 (pThr389) was assessed by western blot to confirm inhibition of mTORC1 signalling. β-actin was used as a loading control. ( D, F, H, J ) Activation of autophagy was quantified by densitometry of LC3B-II normalised to β-actin. Data was pooled from at least three independent experiments and error bars = ±S.E.M. * p< 0.05, ** p< 0.01 by two-way ANOVA test.

Article Snippet: Membranes were incubated overnight at 4 °C on a rocker with the following antibodies in Blocking buffer: β-actin (Sigma-Aldrich, Gillingham, UK A5441), LC3B (Cell Signaling Technology, Leiden, Netherlands 2775), S6K1 (9202), p-S6K1 (9205), ERK1/2 (9102), p-ERK1/2 (9106), ULK1 (4773), p-ULK1 (6888), p-AKT (9271), PTP4A3 (Santa Cruz Biotechnology, Heidelberg, Germany sc-130355), AKT , pan-PTP4A3 (Bio-Techne MAB32191), and GFP (Thermo Fisher Scientific, Horsham, UK A-11122), then washed thrice with TBS-T and incubated with species-appropriate HRP- conjugated secondary antibodies (Thermo Fisher Scientific) diluted in Blocking buffer.

Techniques: Infection, Control, shRNA, Expressing, Western Blot, Incubation, Phospho-proteomics, Inhibition, Activation Assay

Journal: bioRxiv

Article Title: Targeting of PTP4A3 overexpression sensitises HGSOC cells towards chemotherapeutic drugs

doi: 10.1101/2024.10.28.620719

Figure Lengend Snippet: ( A ) Kuramochi-WT, Scr and shPTP4A3 and ( B ) OVCAR 4-WT, Scr and shPTP4A3 cells were treated with increasing concentrations of JMS-053 (iPRL; 0 - 25 µM) for 2 h. Cell extracts were analysed for phosphorylation of ULK1 (pSer757), S6K1 (pThr389), AKT/PKB (pSer473) and ERK1-2 (pThr202-Tyr204). LC3B was used as a marker of autophagy activity, β-actin was used as a loading control, and PTP4A3 knockdown was also confirmed. ( C-F ) The basal phosphorylation of AKT and ERK in WT, Scr and shPTP4A3 cells (no JMS-053 treatment) in ( C, D ) Kuramochi and ( E, F ) OVCAR 4 cells was determined. Data was pooled from three independent experiments and error bars = ±S.E.M.

Article Snippet: Membranes were incubated overnight at 4 °C on a rocker with the following antibodies in Blocking buffer: β-actin (Sigma-Aldrich, Gillingham, UK A5441), LC3B (Cell Signaling Technology, Leiden, Netherlands 2775), S6K1 (9202), p-S6K1 (9205), ERK1/2 (9102), p-ERK1/2 (9106), ULK1 (4773), p-ULK1 (6888), p-AKT (9271), PTP4A3 (Santa Cruz Biotechnology, Heidelberg, Germany sc-130355), AKT , pan-PTP4A3 (Bio-Techne MAB32191), and GFP (Thermo Fisher Scientific, Horsham, UK A-11122), then washed thrice with TBS-T and incubated with species-appropriate HRP- conjugated secondary antibodies (Thermo Fisher Scientific) diluted in Blocking buffer.

Techniques: Phospho-proteomics, Marker, Activity Assay, Control, Knockdown