Lambda protein phosphatase λpp

Cells were stimulated with 50 ng/ml Biotin-TNF (R&D systems) for each indicated time point. For “0” time points, Biotin-TNF was added to cleared cell lysates. Two 15 cm plates of cells at 80% confluency were used for each condition. Media was replaced with 10 mL fresh media prior to lysis to remove unbound TNF. Cells were lysed in buffer containing 30 mM TrisHCl, 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 1% Triton X-100 supplemented with 1 mM DTT, 5 mM N-Ethylmaleimide (NEM; Sigma Aldrich), cOmplete protease inhibitors and phosSTOP. Lysates were incubated on ice for 15 min and cleared by centrifugation. Lysate samples were collected and for ”0” time point samples, Biotin-TNF was added. Lysates were subsequently incubated with Streptavidin magnetic beads (Thermo Fisher Scientific; 88816) for at least 2 h at 4°C and washed five times in lysis buffer. Samples were eluted with 1x LSB for 5 min at 95°C. For phosphatase treatment, beads were resuspended and washed prior to LSB elution in 1x protein metallophosphatase (PMP) buffer (New England Biolabs) supplemented with MnCl₂ and incubated for 30 min at 30°C with lambda protein phosphatase (λPP, New England Biolabs). Samples were analyzed by SDS-PAGE and immunoblotting.

cAMP-dependent protein kinase (PKA) and lambda protein phosphatase (λPP) were purchased from New England Biolabs.

To generate the poly-clonal antibody that specifically recognizes the ADF7 phosphorylated at Ser128, a phosphorylated peptide (ELDGIQVELQATDPSEM(P)SFDIIK) was synthesized and used to immunize rabbit to generate the antibody designated as anti-phospho-ADF7(Ser128). The specificity of the antibody was examined by performing western blot analysis after incubation of ADF7 with CDPK16 in the presence or absence of calcium. To detect the phosphorylated ADF7 in pollen, total proteins were isolated from pollen derived from proADF7::8His-gADF7; adf7 plants. To perform phosphatase treatment, Lambda Protein Phosphatase (λpp, New England Biolabs, P0753S) was added into the extraction buffer. The total protein extract was subsequently incubated with Ni-NTA agarose that was washed extensively with protein extraction buffer after centrifugation. The protein bound to the Ni-NTA agarose was separated by 15% SDS-PAGE and subjected to western blot analysis probed with Anti-phospho-ADF7(Ser128). The relative amount of loaded protein was determined by performing western blot analysis using anti-ADF7 antibody [24 (link)]. The total pollen extract without λpp treatment was used as the control.