Purified FAM122ANterm (~25 µg) and S62 tpARPP19S104A (~25 µg or 125 µg, see preparation in ‘Phosphorylation of ARPP19’ in Methods) alone or in combination were mixed with Expi293F whole-cell extracts expressing B55, PP2Ac constructs and purified PP2Aa. Input samples were collected prior to incubation with agarose beads. GFP-tagged B55 and associated proteins were captured by incubating equal amounts of total protein (500 µg) for each condition with GFP-Trap nanobody agarose beads (prepared using AminoLink Plus Immobilization Kit; ThermoFisher) at 4 °C for 16 h. Following 3 washes with wash buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 1 mM MnCl2), bound proteins were eluted with 2% SDS sample buffer (90 °C, 10 min), resolved by SDS–PAGE (Bio-Rad) and transferred to PVDF membrane for western blot analysis using indicated antibodies (see Reporting summary) anti-FAM122A (MA5-24510, 1:1,000), anti-ARPP19 (Proteintech, 11678-1-AP, 1:1,000). Antibody fluorescence signals were captured using a ChemiDoc MP Imaging System (Image Lab Touch Software 2.4; Bio-Rad) and band intensities quantified using ImageJ 1.53t. Uncropped blots shown in Supplementary Fig. 2 .
Chemidoc mp imaging system image lab touch software 2.4
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The ChemiDoc MP Imaging System is a compact and versatile imaging system designed for a wide range of applications in life science research. It features a high-resolution camera and precise optical system for capturing images of various samples, including gels, blots, and microplates. The system is controlled by the Image Lab Touch Software 2.4, providing a user-friendly interface for image acquisition and analysis.
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3 protocols using chemidoc mp imaging system image lab touch software 2.4
1
Protein Interaction Assay with FAM122A and ARPP19
2
Purified FAM122A Interactome Characterization
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Purified FAM122A and variants (~25 μg, see preparation in methods ) were mixed with Expi293F whole cell extracts expressing B55, PP2Ac constructs and purified PP2Aa. Input samples were collected prior to incubation with agarose beads. GFP-tagged B55 and associated proteins were captured by incubating equal amounts of total protein (~500 μg) for each condition with GFP-Trap nanobody agarose beads (see preparation in methods ) at 4°C for 16 hours. Following 3 washes with wash buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 1 mM MnCl2), bound proteins were eluted with 2% SDS sample buffer (90°C, 10 min), resolved by SDS-PAGE (Bio-Rad) and transferred to PVDF membrane for Western blot analysis using indicated antibodies (see reagents table). Antibody fluorescence signals were captured using a ChemiDoc MP Imaging System (Image Lab Touch Software 2.4; Bio-Rad) and band intensities quantified using ImageJ 1.53t. Uncropped blots shown in Extended Data Fig. 15 .
3
Alkaline Treatment of PP2A:B55
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For alkaline treatment, 100 μl PP2A:B55 triple complex fraction from anion exchange was mixed with NaOH to a final concentration of 0.2 M and incubated for 10 min at RT. The reaction was neutralized by adding HCl to a final concentration of 0.2 M and diluted to 200 μl with lysis buffer. The control reaction was treated with pre-neutralization solution (0.2 M NaOH and 0.2 M HCl) and diluted to 200 μl with lysis buffer. The samples were boiled with 2% SDS sample buffer (90°C, 10 min), resolved by SDS-PAGE gel (Bio-Rad) and transferred to PVDF membrane for Western blot analysis using indicated antibodies (see reagents table). Antibody fluorescence signals were captured using a ChemiDoc MP Imaging System (Image Lab Touch Software 2.4; Bio-Rad) and band intensities quantified using ImageJ 1.53.
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