Determination of phosphorylation sites on CryAB isolated from muscle. a and b) Either endogenous or overexpressed NUAK can form a complex with CryAB as assayed by PLA experiments. a) Maximum intensity projections of L3 muscle tissue (F-actin, lower panels) to visualize the NUAK-CryAB complex (top panels). Scale bar, 25 µm. b) Scatter plot showing an increase in the number of PLA(+) puncta/50 µm² for tagged CryAB with and without NUAK compared to Mef2 > lacZ or PLA controls. One-way ANOVA was performed and the indicated P-values are in comparison to lacZ. N = 10. c) Western blots confirm that the 3xHA-tagged version of CryAB is phosphorylated in muscle tissue. Bands corresponding to phosphorylated CryAB can be visualized with either anti-CryAB (left panel) or anti-HA (right panel) upon expression of Mef2 > CryAB::3xHA alone or with NUAK FL expression. d) Strategy depicting expression and isolation of CryAB for phosphorylation site identification by MS. e) Table with a summary of phospho-MS experiments. Three independent biological replicates were analyzed with total peptide coverage of CryAB ranging from 75.6–78.6%. Serines corresponding to amino acid residues 68 and 70 were both found to be phosphorylated with confidence values >95%. f) Summary of CryAB phosphosites.