The pharmacological inhibitors BIX02189 and WNK463 were obtained from Selleckchem (Houston, TX, USA) and trametinib from LC Laboratories (Woburn, MA, USA). General chemical reagents were purchased from Sigma–Aldrich (St Louis, MO, USA), Merck (Darmstadt, Germany) or BD Biosciences (San Jose, CA, USA).
The antibodies were obtained as follows: anti‐MEK5 was from Enzo Life Sciences (Farmingdale, NY, USA); anti‐GAPDH and anti‐ERK1/2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐pERK1/2, anti‐MEK1/2, anti‐pMEK1/2, anti‐WNK1 and anti‐MEKK2 from Cell Signaling Technologies (Danvers, MA, USA); anti‐Calnexin from Stressgen Bioreagents (Victoria, BC, Canada); anti‐Ki‐67 (clone SP6) from Vitro Master Diagnostica (Granada, Spain); anti‐pWNK1 from R&D Systems (Minneapolis, MN, USA); and horseradish peroxidase‐conjugated secondary antibodies were from Bio‐Rad Laboratories (Hercules, CA, USA). Antibodies raised against ERK5, pERK5 or pMEK5 were developed in our laboratory and have been described.19 , 25 , 26 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).
The antibodies were obtained as follows: anti‐MEK5 was from Enzo Life Sciences (Farmingdale, NY, USA); anti‐GAPDH and anti‐ERK1/2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐pERK1/2, anti‐MEK1/2, anti‐pMEK1/2, anti‐WNK1 and anti‐MEKK2 from Cell Signaling Technologies (Danvers, MA, USA); anti‐Calnexin from Stressgen Bioreagents (Victoria, BC, Canada); anti‐Ki‐67 (clone SP6) from Vitro Master Diagnostica (Granada, Spain); anti‐pWNK1 from R&D Systems (Minneapolis, MN, USA); and horseradish peroxidase‐conjugated secondary antibodies were from Bio‐Rad Laboratories (Hercules, CA, USA). Antibodies raised against ERK5, pERK5 or pMEK5 were developed in our laboratory and have been described.