Fig 2: Immunoblot detection of phosphorylated pHvMPK6 and HvMPK6 in the Hvmpk6 mutant (-1C/-1C) and WT (WT/WT) non-transgenic endosperms with bran and husk. Two representative independent experiments (first experiment and second experiment) are shown. The whole immunoblotted membranes were probed first with an anti-pERK 1/2 antibody and subsequently restriped with an anti-AtMPK6 antibody. For loading control, the whole blotted membranes were stained with Ponceau S. Selected area of the membranes is shown. Mature T3-generation grains of heterozygous mutant (WT/-1C) transgenic plants and mature grains of WT (WT/WT) non-transgenic control plants were imbibed in Milli-Q water at 21°C in darkness for 24 h before embryo extirpation and subsequent analysis. Following embryo extirpation, endosperms with bran and husk were immediately frozen [basal phosphorylation of mitogen-activated protein kinases (MAPKs), mock control] or immediately squeezed and incubated at room temperature for 15 min before freezing (phosphorylated MAPKs, wounding). -1C/-1C endosperms with bran and husk were identified based on the abnormal phenotype of the respective extirpated embryos and subsequent PCR-based genotyping of these embryos. Samples from in vitro grown wounded seedlings of Arabidopsis Col-0 ecotype (Col-0) and mpk6-2 mutant (mpk6-2) were used as references. The red arrowhead indicates the phosphorylated Arabidopsis pMPK6 (anti-pERK 1/2) that is present in Col-0 but missing in mpk6-2, and the green arrowhead indicates Arabidopsis MPK6 (anti-AtMPK6), which is present in Col-0 but missing in mpk6-2. Light blue arrowhead indicates another phosphorylated Arabidopsis pMPK (anti-pERK 1/2), most probably pMPK3, as it is more abundant in mpk6-2 than in Col-0 possibly to partially compensate for the absence of MPK6 (Genenncher et al., 2016). The red arrow indicates phosphorylated barley pHvMPK6 (anti-pERK 1/2), which is present in WT/WT but missing in the -1C/-1C endosperms with bran and husk and the green arrow indicates barley HvMPK6 (anti-AtMPK6), which is present in WT/WT but missing in the -1C/-1C endosperms with bran and husk. The light blue arrow indicates another phosphorylated barley pMPK (anti-pERK 1/2), possibly barley pMPK3 (pHvMPK3), as it is detected only in wound treated -1C/-1C endosperms with bran and husk but missing in the mock-treated mutant -1C/-1C endosperms with bran and husk. Similar to Arabidopsis pMPK3, a higher phosphorylation level of pHvMPK3 may partially compensate for the absence of HvMPK6. The gray arrows indicate two unknown proteins (anti-pERK1/2), which are highly phosphorylated and/or abundant in WT/WT endosperms with bran and husk irrespectively of the treatment conditions (mock and wounding) but reduced or missing in -1C/-1C endosperms with bran and husk. L-Precision Plus Protein™ Dual Color Standards (#1610374, BioRad, Hercules, CA, USA). Sizes of the selected ladder fragments are shown on the left of the membranes. Full scans of the whole immunoblotting membranes are shown in Supplementary Figures 11, 12.

Fig 3: Following purification of the recombinant AAEL002675 protein. Proteins were resolved via SDS-PAGE and probed for the presence of a V5 tag on the protein of interest. Recombinant AAEL002675 (43 kDa) was detected in the first elution fraction from the protein purification process. Lane 1: Precision Plus Protein Dual Color Standards™ (Bio-Rad 1610374); lane 2: S2 supernatant HisPur column flow-through; lane 3: elution fraction 3; lane 4: elution fraction 2; lane 5: elution fraction 1.

Fig 4: T387P decreases hEAAT1 transporter density in the surface membrane. (a) Representative confocal images from HEK293T cells expressing WT (left) or T387P (right) hEAAT1-YFP fusion proteins. (b) YFP-fluorescence distributions (grey values, a. u.) from WT (grey circles, black line, n = 72) and mutant (red squares, red line, n = 24) YFP-fusion proteins from HEK293T cells. Linear fits to the values point to slightly different surface insertion ratios for WT and mutant EAAT1 (slopesWT/T387P: 0.84/0.71; R 2 WT/T387P: 0.97/0.91) (c) Representative cropped region of a SDS-PAGE showing YFP-fluorescence from total lysate (lys) or surface biotinylated protein (surf) for WT and T387P hEAAT1-YFP. MWL = Molecular Weight Ladder, BioRad-Precision plus, Dual color, #1610374. The corresponding full-length gel is provided as Supplementary Fig. 3. (d) Pooled total YFP- fluorescence emissions from WT (circles) and T387P hEAAT1-YFP (squares, n = 5/5). (e) Statistical analysis of surface biotinylation from experiments as shown in c indicates a lower ratio for surface expression of expressed protein for the mutant (P t = 0.011, n = 5/5). (f) Surface insertion probability for core- and complex-glycosylated WT (black bars) and T387P (grey bars) protein.

Fig 5: MIA PaCa-2 pancreatic cancer cells morphology is affected by PGRMC1 phosphorylation status. a PGRMC1-HA proteins constructed for this figure. TMH: Trans-membrane helix. HA: the C-terminal 3x hemaglutinin tag. b Detection of exogenous PGRMC1 expression levels by western blot (upper panel). Equal loading is controlled by quantifying beta actin (lower panel). The results show three totally independent stably transfected cell lines per plasmid from (A). Open arrow: Exogenous PGRMC1-HA (Ex.). Shaded arrow: endogenous PGRMC1 (End.). Filled arrow: beta actin. The molecular weight ladder is Bio-Rad 1610377 Dual Xtra Standards. c Box plots quantification of replicate gels of (B) with signals normalized to beta actin from the same respective lanes. n = 4 lanes for MP and n = 6 for WT, DM and TM (replicates of respective lines 1–3 per condition). There were no significant differences (ns) except for the exogenous band in MP (ANOVA, post-hoc Dunnet’s T3). d Western blot quantification of HA-tagged exogenous PGRMC1, following B but detecting PGRMC1 with anti-HA antibody. The molecular weight ladder is Abcam ab116028 Prestained Protein Ladder. e PGRMC1 mutant protein expression alters MIA PaCa-2 cell morphology. PGRMC1-HA-expressing stable cells (respective lines 1 from B) or MP cells were stained with a FITC-tagged anti-HA antibody (Anti-HA) and imaged by confocal microscopy. DNA was stained with DAPI. Cells were also imaged in differential interference contrast (DIC) microscopy mode. The respective left panels show merged images of all 3 channels. f The rounded phenotype of double and triple mutant (E) was reversed to elongated phenotype after 125 µM ROCKI addition, but not by addition of DMSO vehicle control