α gfp

Prior to cell wall digestion, Cel1-4xFLAG expression was induced for 24h in CIM at 37°C. The Sec63-GFP expression strain was grown overnight in YPD at 30°C, followed by a 3h growth in SC+250 μM CuSO₄ at 30°C (starting OD 0.4). Cells were harvested (3900 rpm, 5 min) and washed once with 0.1 M Tris/HCl pH9. Cells were then resuspended in DTT-buffer (0.1 M Tris/HCl, 10 mM DTT, pH 9.0) and incubated for 20 min at 30°C and 70 rpm. After DTT incubation, cells were washed with Spheroplast buffer (0.1 M Tris, 1.1 M Sorbitol, pH 7.4) once. Cells were resuspended in spheroplast buffer and aliquoted into 5 mL aliquots. To start cell wall digestion Zymolyase yeast lytic enzyme (ZymoResearch) was added to the cells (concentration range: 0 to 200 units), and cells were incubated for 3h at 30°C and 70 rpm. Then, cells were harvested (2200x g 8 min), the supernatant removed and the pellet resuspended in PBS and a TCA based protein extraction was performed as described earlier. Cel1-4xFLAG protein levels were analyzed via western blotting as described earlier. Sec63-GFP protein levels were analyzed using a mouse anti-GFP in a dilution of 1:1000 (α-GFP, ROCHE), followed by the incubation with an anti-mouse-HRP secondary antibody in a dilution of 1:4000 (GE Healthcare).

For western blotting, samples were sonicated for 5 min and boiled in a solution of 2× Laemmli Sample Buffer and 10% β-mercaptoethanol before being fractionated on a 4–12% NuPAGE Bis-Tris gel (Invitrogen). The iBlot Gel Transfer system (Invitrogen) was then used to transfer samples to a nitrocellulose membrane. The following antibodies were used at 1:3000–10,000 dilutions: α-Tubulin: α-Tubulin (DM1a; Sigma), α-GFP: IgG₁κ (Roche), α-SAS-5 (Song et al. 2011 (link)), and α-TBG-1 (Stubenvoll et al. 2016 (link)). IRDye secondary antibodies (LI-COR Biosciences, Lincoln, NE) were used at a 1:10,000 dilution. Blots were imaged using the Odyssey infrared scanner (LI-COR Biosciences), and analyzed using Image Studio software (LI-COR Biosciences).

Parasites were obtained by lysis of the RBC membrane using 0.15% saponin/PBS and incubation on ice for 10 min. The obtained parasite pellet was washed 2–3 times in ice-cold PBS. The whole cell protein extract was generated by lysing the parasite pellet in an equal volume of UREA/SDS buffer (8 M Urea, 5% SDS, 50 mM Bis–Tris, 2 mM EDTA, 25 mM HCl, pH 6.5) supplemented with 1 mM DTT and 1 × protease inhibitor cocktail (Merck). For each sample, protein lysate derived from equal parasite numbers were separated on a 3–8% NuPage Tris–Acetate gel using NuPage MES buffer (Novex, Qiagen). The membrane was blocked in 5% milk powder dissolved in 1xPBS/0.1% Tween (PBS-Tween) for 30 min. Proteins were detected using the primary antibodies mouse mAb α-GFP (1:1,000) (Roche Diagnostics #11814460001), or the mouse mAb α-PfGAPDH (1:20,000)^{65 (link)}, diluted in blocking buffer. The membrane was incubated at 4 °C over night and subsequently washed 3–4 times using PBS-Tween. The secondary antibody α-mouse IgG (H&L)-HRP (GE healthcare #NXA931) was diluted 1:10,000 in blocking buffer and the membrane was incubated for 1–2 h and subsequently washed again 3–4 times using PBS-Tween.