Protein Disulfide-Isomerases

To determine how the adoption of different reference genes can affect the normalization of the expression data for a gene of interest, the same 24 plant cDNA samples used for the stability analyses of reference genes (see Plant material) were also analysed by qRT-PCR for the expression of TaPDIL1-1 (a gene encoding the Protein Disulfide Isomerase). Isolation and characterization in wheat of the three homoeologous gene sequences encoding PDI (TaPDIL1-1) and of their full-length transcripts have been reported previously [40 (link)]. The primer pair used for TaPDIL1-1 qRT-PCR analysis (forward: 5'-CGTGGTCTTCAAATCCTG-3'; reverse 5'-GTAACCCTGGACATCAAAC-3') was designed in conserved regions of the three homoeologous cDNA sequences at their 3' ends (Accession numbers: AJ868105, AJ868106, AJ868107) with annealing temperature of 55°C. The PCR efficiency was 100.35 ± 0,365 with a coefficient of determination (R²) of 0,998; the amplicon size was 187 bp. The TaPDIL1-1 expressions were normalized using five different strategies: 1) geometric average of the three references genes selected as the most stable genes by geNorm; 2) geometric average of the two references genes selected as the most stable genes by NormFinder; 3) the single most stable gene identified by NormFinder; 4–5) the two genes related to the Unigene clusters Ta54825 (Actin) and Ta25534 (α-tubulin) used alone. Normalized TaPDIL1-1 relative values are given as mean value ± SD. Standard deviations on normalized expression levels were computed according to the geNorm user manual [47 ].

The HAI-2 mAb DC16 [9 ] and XY9 were generated by hybridoma fusion of cells isolated from the spleen of a mouse immunized with soluble recombinant HAI-2 (Novoprotein, Summit NJ) as the previously described [9 ]. The mouse monoclonal antibodies M24, M69, and M19 were used for immunoblot analyses to detect total matriptase, activated matriptase and HAI-1, respectively as we have previously described [17 (link)–19 (link)]. The HAI-1 mAb M19 and HAI-2 mAb DC16 were immobilized on Sepharose 4B at 5mg/ml beads, using CNBr-activated beads, following the manufacturer’s instruction and were used for the immunodepletion experiments. The rabbit polyclonal antibody directed against protein disulfide isomerase was obtained from Stressgen (Catalog number SPA-890, Victoria, BC, Canada). FITC-labelled mouse anti-GM130 monoclonal antibody was purchased from BD Bioscience Pharmingen (Catalog number 612009).

Amo knockout flies (amo¹) have been
described [6] (link). Transgenic flies expressing wild type and mutant Amo
(D627V) were generated by BestGene Inc. (USA) using site-specific recombination
with an attP landing site on the second chromosome (Bloomington stock number
9732) [35] (link).
The genomic rescue construct contained the genomic region of
amo, (CG6504) and approximately 1 kb of 5′ flanking
sequence [6] (link).
The mutation in the amo (CG6504) genomic rescue construct was
generated by site-directed mutatgenesis using standard procedures. We sequenced
all constructs to verify that no errors were introduced by PCR. Flies expressing
the wild type and mutant genomic rescue constructs were crossed into the
amo null background for analysis of fertility, sperm
storage and sperm dynamics. Flies expressing Protamine-B-labelled with red
fluorescent protein (ProtB-DsRed) on the third chromosome were kindly provided
by John Belote and a line expressing don-juan-GFP (dj-GFP) on the third
chromosome was obtained from Barbara Wakimoto [20] (link), [36] (link). These lines were
recombined to yield a strain expressing both fluorescent proteins on the same
third chromosome. Flies expressing GFP tagged protein disulfide isomerase were
obtained from Bloomington (stock number 6839). All flies were reared according
to standard procedures and maintained at 25°C.

Mice were sacrificed and transcardially perfused with 4% paraformaldehyde (PFA) in cold phosphate-buffered saline (PBS). Mouse brains, superior cervical ganglions, and adrenal glands were collected, post-fixed in 4% PFA/PBS solution overnight, submerged in 30% sucrose in PBS for at least 72 h, and sectioned at 40 μm thickness using CM1950 cryostat (Leica)^{22 (link)}. Frozen sections were stained with antibodies specific to p150^Glued (amino acid 3–202 at the N-terminus of p150^Glued, BD Biosciences, #610474, 1:200, recognizing p150^Glued but not p135+), p150^Glued & p135+ (amino acid 1266–1278 at the C-terminus of p150^Glued, Abcam, #ab11806, 1:500, recognizing both p150^Glued and p135+), tyrosine hydroxylase (TH, Pel-Freez, #P40101-150, 1:2500; ImmunoStar, #22941, 1:500; Synaptic Systems, #213104, 1:500), dopamine transporter (DAT, Millipore, #MAB369, 1:500), vesicular monoamine transporter 2 (VMAT2, Synaptic Systems, #138302, 1:1000), glial fibrillary acidic protein (GFAP, Abcam, #ab7260, 1:1000), TAR DNA-binding protein 43 (TDP-43, Proteintech, #10782-2-AP, 1:500), α-synuclein (Santa Cruz, #sc-7011-R, 1:500; Santa Cruz, #sc-69977, 1:500), phosphorylated α-synuclein (Ser129) [p-α-synuclein (Ser129), Abcam, #ab51253, 1:500], neuronal nuclei (NeuN, Millipore, #ABN91, 1:500), synaptophysin (Millipore, #AB9272, 1:500), binding immunoglobulin protein (BiP, also referred to as GRP78, Abcam, #ab21685, 1:500), reticulon 3 (RTN3, Proteintech, #12055-2-AP, 1:500), 63 kDa cytoskeleton-linking membrane protein (CLIMP63, Proteintech, #16686-1-AP, 1:500), calnexin (Abcam, #ab22595, 1:500), protein disulfide isomerase (PDI, Proteintech, #11245-1-AP, 1:500), receptor binding cancer antigen expressed on SiSo cells (RCAS1, Cell Signaling Technology, #12290, 1:500), early endosome antigen 1 (EEA1, Cell Signaling Technology, #3288, 1:500), sequestosome 1 (SQSTM1, MBL, #PM066, 1:500), cathepsin D (R&D Systems, #AF1029, 1:500), ER-Golgi intermediate compartment 53 kDa protein (ERGIC53, Sigma-Aldrich, #E1031, 1:500), 130 kDa cis-Golgi matrix protein (GM130, BD Biosciences, #610822, 1:500), phosphorylated eukaryotic translation initiation factor 2α (Ser51) [p-eIF2α (Ser51), Abcam, #ab32157, 1:500], and phosphorylated inositol-requiring enzyme 1α (Ser724) [p-IRE1α (Ser724), Abcam, #ab48187, 1:500] as suggested by manufacturers. Alexa Fluor 488-, 546-, or 647-conjugated secondary antibody (Invitrogen, 1:500) was used to visualize the staining. Fluorescent images were captured using LSM 880 laser-scanning confocal microscope with Zen software (Zeiss) in conventional or Airyscan mode. As a high-resolution imaging modality, the Airyscan technology is reported to improve resolution 2-fold and signal-to-noise ratio 8-fold relative to the conventional confocal microscopy^{61 (link)}. The paired images in all the figures were collected at the same gain and offset settings. Post-collection processing was applied uniformly to all paired images. The images were presented as a single optic layer after acquisition in z-series stack scans at 1.0 μm intervals from individual fields or displayed as maximum-intensity projection or three-dimensional (3D) reconstruction to represent confocal stacks.

In order to validate the transcriptome data, eight selected differentially expressed genes from the RNA-seq analysis [protein disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase (PPI), ubiquitin-protein ligase E3 C (UBE3C), Heat shock protein 70 (Hsp70), zinc finger protein (Msn2), thiol-specific peroxiredoxin (PRDX5), C-5 sterol desaturase (ERG3), delta(9) fatty acid desaturase (OLE1),] were detected by quantitative real-time reverse transcriptase PCR (qRT-PCR). qRT-PCR data were normalized using the P. pastoris glyceraldehyde-3-phosphate dehydrogenase (GAPDH) coding gene as an internal reference gene. Primers were designed using Primer Premier 5.0 software based on the above sequences (Table S4). After 5 days of culture, the total RNA was extracted from each sample using the RNeasy Mini Kit (TransGen Biotech Co., Ltd., Beijing, China) and treated with DNase I to remove genomic DNA contamination, according to the manufacturer’s instructions. The concentrations of purified RNA were determined by measuring the absorbance of samples at 260 and 280 nm, and then first-strand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Internal reference gene and target genes were amplified using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The relative quantification of gene expression was estimated using the comparative 2^−∆∆Ct method, and all reactions were performed in triplicate.