Anti gst antibody

For the pull-down assays, 3 μg bait and prey proteins were incubated overnight at 4°C. The beads were washed with a solution containing 20 mM Tris (pH 7.4), 150 mM NaCl, and 0.05% Tween 20 separated on a sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and analyzed by immunoblotting using an anti-GST antibody (GenScript, Nanjing, China; A00866-100, lot: 18A001413) or an anti-His antibody (Abmart, Shanghai, China; M30111M, lot: 293871). Co-IP was performed as described previously (Su et al., 2017 (link)). Briefly, 1 × 10⁶ protoplasts were lysed with PEN-140 buffer (140 mM NaCl, 2.7 mM KCl, 25 mM Na2HPO4, 1.5 mM KH2PO4, 0.01 mM EDTA and 0.05% CA-630). The supernatant was precipitated with anti-FLAG (Sigma-Aldrich; H8592, lot: SLBV3799) antibodies, followed by washes with PEN-400 buffer (400 mM NaCl, 2.7 mM KCl, 25 mM Na2HPO4, 1.5 mM KH2PO4, 0.01 mM EDTA and 0.05% CA-630). The samples were analyzed by immunoblotting using anti-HA (Roche; 11867423001, lot: 13500600) and anti-FLAG antibodies.

In vitro PARylation assays were performed as described previously (Kong et al., 2021 (link); Yao et al., 2021 (link)). Briefly, 500 ng HIS-FonPARP1 were incubated in a 50 μL PARylation buffer (50 mM Tris–HCl, 50 mM NaCl, 10 mM MgCl₂, pH8.0) with 0.2 mM NAD⁺, 1× activated DNA (BPS Bioscience, San Diego, CA, USA), and 3 μg GST-FonKin4 at 26°C for 3 h. Subsequently, the samples were separated on 8% and 12.5% SDS-PAGE. PARylated proteins were detected via immunoblotting with anti-poly-ADPR antibody (Cat. #MABE1031, Sigma-Aldrich), anti-GST antibody (Cat. #A00865, GenScript) or anti-HIS antibody (Cat. #A00186, GenScript).
For in vitro phosphorylation-mediated self-PARylation assays, 4 μg HIS-FonPARP1 underwent prior in vitro phosphorylation by HIS-FonKin4-ST, as described above. Then, 50 μL PARylation buffer was then added, followed by incubation at 26°C for 30 min. The reaction was stopped by adding 4× SDS loading buffer, and the proteins were then separated on 8% and 12.5% SDS-PAGE. Phosphorylated proteins were detected by anti-phosphor Ser/Thr antibody. PARylated proteins detected through immunoblotting with streptavidin-HRP (Cat. #21126, Thermo Fisher Scientific, Waltham, MA, USA) for Biotin NAD⁺ or anti-HIS antibody (Cat. #A00186, GenScript).

Clarified cell lysate was diluted in denaturing SDS gel loading buffer, and boiled for 15 min. After denaturing, the samples were loaded onto a 4% to 12% gel for SDS-PAGE and separated by electrophoresis. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, ISEQ00010). The PVDF membrane was blocked with 5% skim milk in PBS containing 0.1% Tween 20, and then incubated with the following primary antibodies: anti-Flag antibody (Genscript, A00187), anti-Myc antibody (Genscript, A00172), anti-GST antibody (Genscript, A00097), or anti-TfR1 antibody (Santa Cruz Biotechnology, sc-32272). Then, the PVDF membrane was washed three times with PBS and incubated with HRP-conjugated Goat anti-Mouse antibody (Genscript, A00160) and Goat anti-Rabbit antibody (Genscript, A00098). After three washes with PBST buffer, target protein bands were detected by using the enhanced chemiluminescence (ECL) reagent (Merck Millipore, WBLUR0500).