Complete counting cocktail 3a70b

For metabolic labeling, cells were incubated in methionine/cysteine (Met/Cys)-free DMEM (Corning, 17-204-CL) supplemented with 40 mM HEPES, 2 mM L-glutamine and a ³⁵S-L-methionine and ³⁵S-L-cysteine mix (PerkinElmer, NEG072007MC) for 30 min prior to cell lysis. For each ml of labeling media prepared, 0.035 mCi of the ³⁵S Met/Cys mix was added. Where inhibitors were used, inhibitors were present during labeling. After in-well lysis in Laemmli buffer, samples were resolved by SDS-PAGE and gels were then fixed in 10% acetic acid/25% methanol solution for 30 min. The fixed gels were then dried at 80°C for 2 h using a Model 583 Gel Dryer (Biorad) and exposed to audioradiography film.
To quantify the radioactivity of ³⁵S present in the label samples, 20 μl of radiolabeled sample was incubated with 10 μl of 10 mg/ml BSA and 1 mL of ice-cold 10% TCA solution for 30 min on ice. Precipitated proteins were vacuum filtered using a 1225 Sampling Manifold (Millipore Sigma) onto glass microfiber filters (GE Life Sciences, 1822-025), and washed twice each with ice-cold 10% TCA solution and 95% ethanol. Filter counting was performed by immersing the filters into 3 mL of Complete Counting Cocktail 3a70B (Research Products International Corp., 111154). The number of counts registered per minute (CPM) was measured using a Beckman LS 6500 liquid scintillation counter with a counting time of 5 min.

WNK1-KDm/S*, p38α, MEK6, or ASK-KDm, or the WNK1 mutants
was added at a final concentration of 2 μM to 10 molar excess of MBP in
30 μl of kinase reaction buffer (30 mM HEPES pH 8.0, 5 mM
MgCl₂, 1 mM DTT, 100 μM ATP) containing 10 μCi of
[γ32P]-ATP. Increasing amounts of NaCl were added to the
reaction mixture to make a series of final chloride concentrations in the range
of 10–1000 mM. Reactions were incubated for 1 hour at 37 °C.
Reactions were stopped by blotting 20 μL of each reaction on filter
paper squares (1 cm × 1 cm) and plunging into a solution of 10%
trichloroacetic acid (TCA). The squares were washed three times in TCA, blotted,
and transferred to scintillation vials. The vials were filled with 5 ml Complete
Counting Cocktail 3a70B (Research Products International Corp.) and counted on a
Beckman LS 3801. Reactions were normalized to 100% highest signal and
0% for the sample lacking substrate (MBP). To confirm that all counts
were the result of MBP phosphorylation, autophosphorylation was monitored by
separation of the reaction mixture on an SDS-PAGE gel followed by scintillation
counting of the band containing WNK1-KDm/S*. No autophosphorylation of
the WNK1 kinase domain was detected in the MBP substrate assay. Mutants were
assayed similarly.

WNK1-KDm/S* was added at a final concentration of 2 μM
to 5 molar excess of OSR1 1–295 in 30 μl of kinase reaction
buffer (30 mM HEPES pH 8.0, 5 mM MgCl₂, 1 mM DTT, 100 μM ATP)
containing 10 μCi of [γ32P]-ATP. Increasing
amounts of NaCl were added to the reaction mixture to make a series of final
chloride concentrations in the range of 30–1000 mM. Reactions were
incubated for 1 hour at 37 °C. Reactions were stopped by blotting 20
μL of each reaction on filter paper squares (1 cm × 1 cm) and
plunging into a solution of 10% trichloroacetic acid (TCA). The squares
were washed three times in TCA, blotted, and transferred to scintillation vials.
The vials were filled with 5 ml Complete Counting Cocktail 3a70B (Research
Products International Corp.) and counted on a Beckman LS 3801. Reactions were
normalized to 100% highest signal and 0% for the sample lacking
substrate (OSR1 1–295).