= Solution
Use a total-<protein> stain or validated constitutive loading control, and measure total amounts of the candidate signaling <proteins> alongside their site-specific phosphorylated forms. This distinguishes increased phosphorylation fraction from increased protein abundance. Include a no-primary or secondary-only control and a phosphatase-treated aliquot to test phospho-dependent detection. \b[Confirm candidate identities using validated target-specific phospho-<antibodies>], suitable positive controls and, where feasible, depletion or inhibition of the relevant target. Repeat the paired time course with biological replicates and non-saturated imaging. Matching culture state, stimulus dose and sampling time is essential because an ordinary difference in response kinetics could otherwise mimic constitutive activation.
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