Genegnome

Protein samples were lysed in RIPA buffer (Beyotime) supplemented with 1% proteinase inhibitors (Beyotime). The protein concentration was determined using a bicinchoninic acid (BCA) assay kit (Beyotime). Equal amounts of proteins (10 µg) were separated on an 8% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel and transferred onto polyvinylidene fluoride (PVDF; Millipore) membranes. The membranes were then blocked in rapid blocking solution and incubated with the primary antibodies overnight at 4°C. The following antibodies were used: CD4 (1:1,000), CD8 (1:1,000), CD3(1:1,000), CX3CL1(1:1000, GAPDH (1:1,000). Finally, the membranes were incubated with HRP−conjugated secondary antibody (1:1,000) for 2 h at room temperature. The immunoblots were visualized using the Immobilon Western Chemiluminescent HRP Substrate, and the bands were quantified, relative to GAPDH, by densitometric analysis (GeneGnome, Syngene UK).

Cell lysates from cells cultured on polyacrylamide gels were prepared with radio immunoprecipitation assay (RIPA) buffer (89900, Thermo-Fisher) and a protease inhibitor cocktail (78440, Thermo-Fisher) for 10 minutes on ice. Lysates were sonicated and clarified by centrifugation at 9600 × G, at 4 °C for 10 minutes. Protein concentration was determined by DC protein assay (500-0116, Bio-Rad). Samples were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. Membranes were blocked with 5% Bovine Serum albumin (BSA, A8022, Sigma) and 0.1% Tween-20(P1379, Sigma) in PBS for 30 minutes. Primary antibodies were prepared in blocking solution and incubated overnight at 4 °C (MMP-2 – sc-10736, Santa Cruz, MMP-9 – sc-10737, Santa Cruz, Timp-1 – sc-5538, Santa Cruz, HSC70 – sc-7298, Santa Cruz). The membrane was washed three times in 0.1% Tween-20/PBS and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies for 1 hour. Following three washes in 0.1% Tween-20/PBS, membranes were developed using Luminata crescendo HRP substrate (WBLUR0100, Millipore) and Syngene GeneGnome. Band intensities were analysed via the band densitometry plugin in ImageJ.

Total protein concentration in the samples was determined using a BCA protein assay kit (Thermo Scientific). Samples were then prepared with loading buffer and denatured by incubating samples at 100°C for 5 min. For western blot analyses, 30 μg total protein was loaded on bis-acrylamide gels and separated using polyacrylamide gel electrophoresis (PAGE). Samples were then transferred to PVDF membrane (Millipore) and blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline (pH 8.0) for 1 h. The appropriate primary antibodies were added: Grp78/BiP (BD Biosciences) (PERK, phospho-PERK (Thr980), IRE1α, eIF2α, phospho-eIF2α (Ser51), PDI, and CHOP; 1:1000, Cell Signaling Technology) and membranes were incubated at 4°C overnight, washed and then and subsequently probed with HRP-linked anti-rabbit IgG or anti-mouse IgG antibodies (1:1000, Cell Signaling Technology) 1 h at room temperature. Secondary antibodies were detected using HRP-linked chemiluminescence with SuperSignal West Dura Chemiluminescence Substrate (Thermo Scientific) and imaged using the chemiluminescence imaging system (GeneGnome, Syngene). The signal for the target protein of each sample was quantified using densitometry (Image J Software) and expressed in arbitrary unit (AU). GAPDH (1:2000, Thermo Scientific) or β-actin (1:1000, Cell Signaling Technology) was used to confirm equal protein loading across samples.

SDS-PAGE and western blot analysis of plant produced Pfs48/45 and Pfs48/45-10C were performed as described previously^{24 (link)}. The FLAG tagged Pfs48/45-10C and Pfs48/45 variants were detected using anti-FLAG mAbs (Cat. No. 637301, BioLegend) or the anti-Pfs48/45 mAb (MRA-316) antibody. Native PAGE of FLAG tagged Pfs48/45 or Pfs48/45-10C variants is performed using native samples and running buffers without denaturants or SDS as described previously^{24 (link)}. The images of the protein bands were taken using the GeneSnap software on a GeneGnome and were quantified using the Gene Tools software (Syngene Bioimaging, UK).

For protein extraction, cells were grown in synthetic media until late logarithmic phase (cell density about 10⁷ cells/ml) and collected with centrifugation (about 10⁷ cells for alkaline lysis [64 (link)] or about 2×10⁸ cells for mechanical cell disruption [65 (link)]). As alkaline lysis allows to achieve higher protein concentration [64 (link)] and reduced degradation (compare S2A and S2B Fig), but due to the procedure requires sample boiling, it was used to check protein production, while mechanical cell disruption was used to assess protein aggregation.
SDS-PAGE [57 ] or SDD-AGE [27 (link)] was used for separation of the proteins, and PVDF membranes (GE Healthcare) were used for semi-dry [57 ] or capillary transfer [66 ], respectively. Blots were probed with either anti-732-743-Sch9 and anti-phospho-Thr737-Sch9 [67 (link)] or anti-Tag(CGY)FP (Evrogen AB121) antibodies and photographed with GeneGnome (SynGene).

To confirm the array data findings, and to examine other proteins of interest, samples were examined for total and phosphorylated protein expression using protein immuno-blotting (Western Blotting, WB) using a standard protocol. Details of the methodology are shown in Supplementary material 2. All antibodies used for WB are shown in Supplementary Table 2. We used the total expression of each individual phospho experiment as the loading control, as per previous work. In the experiments where we had no totals, β-actin was used as a loading control. In terms of exposure times, we used an automated system (GeneGnome, Syngene, Cambridge, UK) that set the optimal exposure timepoints; it was not something that could be edited by us.