anti perk5 antibody  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: A stepwise and digital pattern of RSK phosphorylation determines the outcome of thymic selection

doi: 10.1016/j.isci.2023.107552

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Article Snippet: Anti-pERK5 (1.T218/Y220) , Santa Cruz biotechnology , Cat# sc-135760 RRID: AB_2250338.

Techniques: Bioprocessing, Recombinant, Binding Assay, Mass Spectrometry, Software, Imaging

Journal: Clinical and Translational Medicine

Article Title: The WNK1–ERK5 route plays a pathophysiological role in ovarian cancer and limits therapeutic efficacy of trametinib

doi: 10.1002/ctm2.1217

Figure Lengend Snippet: Anti‐proliferative effect of WNK1–ERK5 axis knockdown. (A) Cells were infected with a pLKO shControl sequence or two different WNK1 specific shRNA sequences. 70 μg of cell extracts were used to determine the WNK1 knockdown by Western blotting. Calnexin was used as loading control. Dashed line indicates that lanes were cut out from immunoblots. (B) The effect of WNK1 knockdown (sh18 and sh21 or sh86) on cell proliferation was measured at 3 days of culture by the MTT assay. Data are presented as the mean ± SD of an experiment that was repeated three times. *, p ≤ .05; ***, p ≤ .001. (C) 70 μg of whole‐cell lysates were used to detect MEKK2 and MEK5 expression in the four ovarian cancer cell lines by Western blotting with their corresponding antibodies. ERK5 activation was analysed by immunoprecipitating 1 mg of protein followed by Western blotting with the anti‐ERK5 or anti‐pERK5 antibodies. Calnexin was used as loading control. (D) OVCAR8 cells infected with pLKO shControl or sh18 were lysed and protein levels were evaluated on 70 micrograms of cell extracts by Western blotting with their corresponding antibodies. ERK5 and pMEK5 were analysed by immunoprecipitating 1 mg of protein followed by Western blotting with anti‐ERK5 or anti‐pMEK5. Calnexin was used as loading control. (E) Knockdown of MEKK2, (F) MEK5 or (G) ERK5 was carried out by lentiviral infection with specific shRNA sequences. MEKK2, MEK5 (70 μg of cell extracts) and ERK5 (1 mg of immunoprecipitated protein) were analysed by Western blotting with the appropriate antibodies. Calnexin was used as loading control. (H) The effect of MEKK2, MEK5 and ERK5 knockdown on cell proliferation was measured at 3 days of culture by the MTT assay. Data are presented as the mean ± SD of an experiment that was repeated three times. **, p ≤ .01; ***, p ≤ .001.

Article Snippet: Antibodies raised against ERK5, pERK5 or pMEK5 were developed in our laboratory and have been described., , The pERK1/2 antibody conjugated to PE fluorophore was obtained from Biolegend (San Diego, CA, USA) and the anti‐pERK5 conjugated to AF488 from Santa Cruz Biotechnology (p‐ERK 5 Antibody (1.T218/Y220) Alexa Fluor ® 488, sc‐135760 AF488).

Techniques: Knockdown, Infection, Sequencing, shRNA, Western Blot, Control, MTT Assay, Expressing, Activation Assay, Immunoprecipitation

Journal: Clinical and Translational Medicine

Article Title: The WNK1–ERK5 route plays a pathophysiological role in ovarian cancer and limits therapeutic efficacy of trametinib

doi: 10.1002/ctm2.1217

Figure Lengend Snippet: Impact of pERK1/2 on patient survival. Anti‐tumoural efficacy of double pathway (ERK5 and ERK1/2) blockade. (A) MEK1/2, pMEK1/2, ERK1/2 and pERK1/2 expression were determined by Western blotting. (B) Levels of pERK1/2 in 63 epithelial ovarian cancer samples quantified from immunoblots in Figure using the ImageJ software. The red line represents the threshold value (4 a.u.) selected for sorting the patients into the high or low pERK1/2 expression groups. (C) Kaplan–Meier curve of the above patients with available clinical data ( n = 52), comparing the overall survival of those expressing high levels of pERK1/2 ( n = 19, red line) with those expressing low levels ( n = 33, black line). (D) OVCAR8 cells were treated with trametinib for 4 h and pERK1/2, ERK1/2 and ERK5 expression determined by Western blotting. (E) pERK1/2 and pERK5 levels were quantified from the previous immunoblots (in D) by using the Image Lab software and represented as percentage from control untreated cells. (F) OVCAR8 cells were plated in p6 wells and treated with trametinib and BIX02189, individually and combined, for 3 days. Cell proliferation, measured by cell counting, was represented as percentage from OVCAR8 untreated cells. Data are presented as the mean ± SD of an experiment that was repeated three times. ***, p ≤ .001. (G) OVCAR8 scramble cells and OVCAR8 MEK5 CRISPR clones (#16 and #19) were plated in 24‐well dishes, and 24 h later treated with the indicated doses of trametinib for 48 h. Cell proliferation was measured by an MTT assay, and each condition was represented as percentage from their respective untreated cells. Results are expressed as mean ± SD of an experiment that was repeated twice. ***, p ≤ .001. (H) Mice xenografted with OVCAR8 Sc or MEK5 CRISPR cells were divided into two groups once they reached an initial mean volume of approximately 500 mm 3 . Each of them received 100 μL of trametinib (0.5 mg/kg) or vehicle, administered i.p. daily for 5 weeks. Tumour progression of Sc and CRISPR groups were measured weekly. At the end point of the experiment, the tumour volume of the trametinib treated mice were relativised to their corresponding untreated (vehicle) groups *, p ≤ .05; **, p ≤ .01; ***, p ≤ .001. (I) Scramble tumours from the vehicle and trametinib treated mice were resected and processed as described in the methods section. pERK1/2 and ERK5 were determined by Western blotting. (J) pERK1/2 (left panel) and pERK5 (right panel) levels from the previous immunoblots (in I) were quantified using the Image Lab software. Comparison of pERK1/2 levels in the scramble tumours treated with vehicle or trametinib was represented as arbitrary units. Comparison of pERK5 expression between groups was represented as percentage of total ERK5 (sum of upper and lower bands). The red lines represent the mean expression values for each group. *, p ≤ .05.

Article Snippet: Antibodies raised against ERK5, pERK5 or pMEK5 were developed in our laboratory and have been described., , The pERK1/2 antibody conjugated to PE fluorophore was obtained from Biolegend (San Diego, CA, USA) and the anti‐pERK5 conjugated to AF488 from Santa Cruz Biotechnology (p‐ERK 5 Antibody (1.T218/Y220) Alexa Fluor ® 488, sc‐135760 AF488).

Techniques: Expressing, Western Blot, Software, Control, Cell Counting, CRISPR, Clone Assay, MTT Assay, Comparison

Journal: Clinical and Translational Medicine

Article Title: The WNK1–ERK5 route plays a pathophysiological role in ovarian cancer and limits therapeutic efficacy of trametinib

doi: 10.1002/ctm2.1217

Figure Lengend Snippet: Anti‐tumoural effect of MEK5 and MEK1/2 targeting in a three‐dimensional model utilising patient biopsies. (A) Ovarian cancer patient tumour biopsies and plasma samples were obtained from ovarian cancer patients. Tissue biopsies were enzymatically digested and grown in HuP3D cultures made from the matching patient plasma. HuP3D cultures were treated with trametinib and BIX02189 and further evaluated by flow cytometry in terms of mean fluorescence intensity (MFI) expression of pERK1/2 and pERK5 as well as survival. (B) Gating strategy for analysis of patient biopsy material grown in HuP3D cultures. Data acquisition was completed by collecting information for a specified number of events determined by counting beads. Firstly, cellular populations were isolated from beads and then singlets were gated. After that, the live cell population was identified by live/dead viability marker. Following this, CD45− BV510 cells were identified from the live cell population. Finally, EpCAM+ (CD326+) PE‐Cy7 cells and FAP− AF700 cells were identified from the CD45− population. Fluorescence minus one (FMO) controls were used to set the gating for each population BV510 (CD45), PE‐Cy7 (EpCAM) and AF700 (FAP). (C) pERK1/2 expression by EpCAM+ cells from ovarian cancer patients (#1–3) growing within HuP3D cultures after DMSO control (Untreated) and trametinib treatment, quantified as MFI ratio between PE‐anti‐pERK1/2 and FMO PE control. Results are expressed as mean ± SD of an experiment that was repeated three times. An interquartile range (IQR) was used to remove outliers. *, p ≤ .05; **, p ≤ .01. (D) Representative flow cytometry histogram of PE pERK1/2 expression of EpCAM+ cells from patient #1 after DMSO control (untreated) and trametinib treatment, with PE FMO and activated controls, as negative and positive controls, respectively. Vertical line is included to indicate the pERK1/2 positive population defined by FMO control. (E) pERK5 expression by EpCAM+ cells from ovarian cancer patients (#1–3) growing within HuP3D cultures after DMSO control (untreated) and trametinib treatment, quantified as MFI ratio between AF488‐anti‐pERK5 and AF488 FMO control. Results are expressed as in (C). (F) Representative flow cytometry histogram of pERK5 expression (AF488) of EpCAM+ cells from patient #1 after DMSO control (untreated) and trametinib treatment, with AF488 FMO and activated controls, as negative and positive controls, respectively. Vertical line is included to indicate the pERK5 positive population defined by FMO control. (G) Effect of DMSO control (untreated), trametinib alone or trametinib in combination with BIX02189 on primary ovarian cancer EpCAM+ cell survival in HuP3D cultures from patients #1 to 3. Results are expressed as in (C). *, p ≤ .05; **, p ≤ .01. ***, p ≤ .001. (H) Representative flow cytometry histogram of pERK1/2 (PE) and pERK5 expressions (AF488) of EpCAM+ cells from patient #2 after DMSO control (untreated), trametinib alone or trametinib in combination with BIX02189.

Article Snippet: Antibodies raised against ERK5, pERK5 or pMEK5 were developed in our laboratory and have been described., , The pERK1/2 antibody conjugated to PE fluorophore was obtained from Biolegend (San Diego, CA, USA) and the anti‐pERK5 conjugated to AF488 from Santa Cruz Biotechnology (p‐ERK 5 Antibody (1.T218/Y220) Alexa Fluor ® 488, sc‐135760 AF488).

Techniques: Flow Cytometry, Fluorescence, Expressing, Isolation, Marker, Control