Anti flag

Peptide arrays were synthesised in‐house on cellulose membrane supports via automatic SPOT synthesis using the MultiPep RSi Robot (Intavis, Tübingen, Germany) and 9‐fluorenylmethoxycarbonyl chloride (Fmoc) chemistry. Peptide sequences from DHX9 and PDE4D7 were spotted as 25‐mer peptides shifted by five amino acids, alongside alanine scans, N‐ and C‐terminal truncations and point substitutions to identify crucial amino acids for protein–protein interactions. For identification of PKA phosphorylation of DHX9, peptide arrays were incubated with or without 100 units of active bovine PKA catalytic subunit. For binding between DHX9 and PDE4D7, membranes were overlaid with either purified protein or overexpressing cell lysate. Far western blotting was then conducted with the following antibodies prior to chemiluminescent imaging: anti‐GST 1 : 1000 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc‐138), anti‐phospho‐PKA‐substrate 1 : 1000 (Cell Signalling, Danvers, MA, USA, #9624S), anti‐FLAG 1 : 1000 (ThermoFisher, #PA1‐984B), Goat‐anti‐rabbit HRP 1 : 2000 (Jackson ImmunoResearch, #111‐035‐144), Rabbit Anti Mouse HRP 1 : 2000 (Jackson ImmunoResearch, West Grove, PA, USA, 315‐035‐003).

Transformants were cultured in liquid V8 medium for 3 d then total proteins were extracted using the BestBio Thick-wall microbial protein extraction kit. N-ethyl maleimide (NEM) was added when detecting PsATG8²² . The resulting protein extract was incubated with 25 μL (0.25 mg) anti-Flag, anti-HA, or anti-Myc agarose beads (Thermo Fisher Scientific) for 3 h at 4 °C. The combined agarose beads were then collected by centrifugation and washed three times with pre-cooled wash buffer (50 mM Tris.HCl, 0.15 M NaCl, pH = 7.4). The immunoprecipitates were separated by SDS-PAGE and detected using the corresponding antibodies.

The detailed protocol is provided in [20 (link)]. The antibodies used were Anti-KCTD1 (LS-C260993, LS-Bio, Seattle, WA, USA), Anti-Flag (MA1-91878, Thermo Fisher), anti-IKβα (662402, Biolegend, USA), anti-β-catenin (Sc-7963, SantaCruz), and anti-β-actin (ab11004, Abcam, UK) as loading controls. Briefly, 30 μg of protein extracts from each sample were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in TBS-T (Tris-buffered pH8/0.15% Tween 20) at room temperature for 1 h. Then, they were incubated with primary antibodies diluted in 5% non-fat milk in TBS-T (according to manufacturer instructions) overnight at 4 °C. After three washes in TBS-T, the membranes were incubated with corresponding secondary antibodies for 1 h at room temperature. The signal intensity was visualized using Clarity Max Western ECL Substrate (BioRad cat#1705062) and acquired using the ChemiDoc Imaging system (Bio-Rad, USA). Semi-quantitative densitometric analysis was performed using Image Lab 5.2.1 (Bio-Rad). Protein band normalizations were conducted dividing the signal of the protein of interest to the signal of β-Actin.

To test the reactivity of Pfs25 samples to antibodies against linear and conformational epitopes on native Pfs25, ELISAs were carried out essentially as described [25 (link)]. Recombinantly-expressed Pfs25 (33.3 ng) and Pfs25:Grl1p (100 ng) were incubated overnight on Nunc Maxisorp plates so that wells contained equimolar amounts of the Pfs25 moiety. Plates were washed with PBST and blocked with 2% BSA/PBST for 1 h at RT. Proteins were incubated with either 32F81, 1G2, 4F7, or anti-FLAG (Thermo Fisher) antibodies in a 5-fold dilution series (0.4 μg/ml to 0.000128 μg/ml) in 2% BSA/PBST for 2 h at RT. After washing with PBST, goat anti-mouse HRP (BioRad) diluted 1:4000 in 2% BSA/PBST was added for 1 h at RT. After washing with PBST, plates were developed with 1-Step Ultra TMB reagent (Thermo Scientific).

The following primary antibodies were used: anti-ARF1 (mouse [mo]; Santa Cruz Biotechnology [SCBT]); anti-ARF3 (mo; SCBT); anti-actin (rabbit [rb]; Sigma); anti-acetylated alpha-tubulin (mo; Sigma); anti-alpha-tubulin (mo; Sigma); anti-HSP70 (chicken [ck]; StressMarq); anti-detyrosinated alpha-tubulin (rb; Abcam, Inc.); anti-FLAG (mo; Thermo Fisher); anti-FLAG (rb; Sigma); anti-HA (chicken; Thermo Fisher); anti-pan-ARF (mo; Millipore); anti-ARF6 (rb; Cell Signaling); anti-ARF5 (mo; Abnova); anti-ARF4 (rb; ProteinTech); anti-giantin (rb; BioLegend); anti-GM130 (mo; Becton Dickinson [BD]); anti-lipopolysaccharide (anti-LPS; mo; Virostat); anti-CT813 (rb; T. Hackstadt); anti-IncA (rb; T. Hackstadt). Goat anti-rabbit and anti-mouse IgG–Alexa Fluor488, -555, and or -647–conjugated secondary antibodies, goat anti-chicken IgY Alexa Fluor555-conjugated secondary antibody, and donkey anti-rabbit and anti-mouse IgG–horseradish peroxidase (HRP)–conjugated secondary antibodies were purchased from Invitrogen. Donkey anti-chicken IgY–HRP–conjugated secondary antibody was purchased from Pierce.