A7470

Four-week-old N. benthamiana leaves were infiltrated with A. tumefaciens cultures carrying the pCambia1300 constructs. Forty-eight hours post infiltration, the leaves were collected and ground in liquid nitrogen. Total proteins were extracted from 2 g of ground powder in 4 ml of IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 2 mM EDTA, 0.5% Triton X-100, 5% glycerol, 5 mM DTT, 1 mM PMSF, and 1 Protease Inhibitor Cocktail tablet (Roche, 4693116001)). The extract was centrifuged three times at 20,000 × g for 10 min each time at 4 °C and the supernatant was collected. Twenty-five microliters of anti-c-myc-agarose affinity gel (Sigma–Aldrich, A7470) or GBP beads (homemade) were added to the supernatant and incubated at 4 °C for 2 h with rotation. After incubation, the beads were washed three times with IP buffer at 4 °C. Finally, 50 μl of 2 × SDS loading buffer were added to the sample for western blot analyses.

HEK293FT cells were transfected with SFB (a triple-epitope tag containing S-protein, FLAG, and streptavidin-binding peptide)-tagged Nfatc1 (full-length or truncation mutants) and MYC-tagged Tead3 (full-length or truncation mutants) and harvested 2 days after transfection. Cells were lysed in RIPA lysis buffer (Sigma, 20-188) at 4 °C for 15 min and sonicated. The lysates were centrifuged at 14,000 rpm at 4 °C for 15 min, and the supernatant was incubated with specific beads or antibodies. For the pulldown of SFB-tagged proteins, cell extracts were incubated with S-protein beads (EMD Millipore, 69704-3). For immunoprecipitation of MYC-tagged proteins, cell extracts were incubated with anti-MYC beads (Sigma, A7470, RRID: AB_10109522). After incubation at 4 °C overnight, the immune complexes were centrifuged and washed with PBS three times, and the bound proteins were eluted by boiling in Laemmli buffer at 95 °C for 10 min, followed by Western blot analysis with the indicated antibodies.

Cells were lysed in an appropriate volume of IP lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100 (v/v), 10% glycerol (v/v), 2 mM EDTA, 25 mM NaF and 2 mM NaH₂PO₄ containing 1% protease inhibitor cocktail (P8340, Sigma-Aldrich) and 1% phosphatase inhibitor cocktails 2 and 3 (P5726 and P0044, Sigma-Aldrich) added fresh] or RIPA buffer [25 mM Tris (pH 7.5), 150 mM NaCl, 0.1% SDS (v/v), 0.5% sodium deoxycholate (v/v), 1% Triton X-100, containing 1 EDTA-free protease inhibitor tablet (Roche), and 1% phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich) added fresh] for 10 min on ice, and proteins were resolved by SDS-PAGE for WB.
For immunoprecipitation of myc-tagged PLK4, lysates were incubated with 50 μl of Myc-tag beads [A7470, Sigma-Aldrich, blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature], for 2 h at 4°C with rotation. The beads were subsequently washed with lysis buffer and eluted with 2× SDS-PAGE sample buffer (Nupage, Invitrogen). Other immunoprecipitation experiments were performed as described previously (Whalley et al., 2015 (link)).