Pka colorimetric activity kit

The activity of protein kinase A (PKA) was assessed utilizing a PKA Colorimetric Activity Kit (Invitrogen), adhering to the manufacturer’s guidelines. Following the assay procedure, the colorimetric measurements were taken at a wavelength of 450 nm using the microplate reader.

PKA activity in HEK293 cells was determined using the PKA Colorimetric Activity Kit (EIAPKA; Invitrogen), following the manufacturer’s instructions. Briefly, cells were cultured in 24-well plates and then treated with forskolin or IBMX for indicated concentrations and time points. Following the stimulation, cells were harvested, and cell lysates were prepared. Substrate phosphorylation was determined via a colorimetric assay and the absorbance was measured at 450 nm using an Infinite M200 microplate reader (Tecan, Männedorf, Switzerland). PKA activity was assessed by calculating the units of kinase activity for each protein sample, which were then normalized to the levels observed in the untreated control samples.

Cells were starved in FBS-free DMEM for 4 h, treated with 100 nM glucagon, 10 μM forskolin, or 100 µM Bt2-cAMP for 30 min, and then lysed in cell lysis buffer (tris-based, pH 8 buffer with 1% NP-40) containing protease-inhibitors and phosphatase-inhibitors cocktail (Sigma). PKA activity was determined with a PKA Colorimetric Activity Kit (Invitrogen, #EIAPKA) as directed by the manufacturer’s protocol.

The effect of methionine sulfoximine (MSO; Sigma; #M5379) on PKA (EMD Millipore; #539481) activity was measured using the PKA Colorimetric Activity Kit (Invitrogen; #EIAPKA) according to the manufacturer’s protocol.

PKA activity was determined in the lenses of 4 months old WT, Cx50KO, Cx46KO, or dKO mice. The lenses were treated with or without 50 μM forskolin or 2 mM 8-Br-cAMP in the absence or presence of H₂O₂ or UVB. Briefly, lenses were homogenized with lysis buffer (0.1% protease inhibitor cocktail, 1 mM PMSF, 10 mM Na₃VO₄) for 30 min on ice with occasional gentle vertexing and then centrifuged at 9200 g for 10 min at 4°C. The pellet was resuspended in 1X Kinase Reaction Buffer from the PKA Colorimetric Activity Kit (EIAPKA, Invitrogen, Frederick, USA) and followed by incubation with PKA standards from the Kit or resuspended pellet samples, and reconstituted ATP in sequence. The sample-containing plates were sealed and incubated for 90 min at 30°C with shaking, and then washed three times with wash buffer from the Kit at room temperature (RT). The goat anti-rabbit IgG HRP conjugated antibody and the phosphor-PKA substrate antibody from the Kit were then added in sequence, and the plates were incubated for 60 min at RT with shaking. The plates were rinsed three times with wash buffer from the Kit at RT, and incubated with the tetramethylbenzidine substrate from the Kit, for 30 min at RT. The stop solution from the Kit was added, and the absorbance at 450 nm was measured with a microtiter plate reader (Synergy HT, Biotek, Winooski, USA).

To quantify the activity of cAMP-dependent PKA [45 (link)], a PKA Colorimetric Activity kit (Thermo Fisher Scientific, Waltham, MA, USA) was used. The cells were seeded in a 6-well plate at a density of 5 × 10⁵ cells/well in MM. After 24 h, the cells were treated with or without glucagon in the presence or absence of 10 µmol/L H89, PKA inhibitor [46 (link)], in MM. After 30 min, the cells were incubated with a cell lysis buffer supplied by the manufacturer for another 30 min on ice with occasional vortexing. After 10 min of centrifugation, the supernatant was collected and used to perform a PKA assay according to the manufacturer’s instructions.