Journal: Cellular Logistics

Article Title: Interaction with the effector dynamin-related protein 1 (Drp1) is an ancient function of Rab32 subfamily proteins

doi: 10.4161/21592799.2014.986399

Figure Lengend Snippet: Localization of Rab32 Family Proteins to the ER, Mitochondria and Lysosomes. A-C. HeLa cells were processed for confocal immunofluorescence microscopy for Rab32 family proteins and the ER (detected with ERp57, ( A ), mitochondria (visualized with Mitotracker, ( B ) and lysosomes (stained with LAMP-1, ( C ). Yellow color in the overlap indicates apposition of the Rab and ER or mitochondria staining. Pictures correspond to extended focus images. Scale bar = 25 μm. Representative images are shown. ( D). Co-localization was quantified using the Manders coefficient for the localization of Rabs with the ER, mitochondria and lysosomes, as shown by representative images in panels A-C. P a = 0.0011, P b = 0.0064, P c = 0.0001, P d = 0.0004. (Standard deviation is shown, n = 6) (E). Rab32 family distribution between microsomes (containing ER, endosomes and lysosomes), mitochondria and the MAM. HeLa homogenates were fractionated via the Percoll fractionation protocol into cytosol, microsomes, pure mitochondria, and MAM, as indicated on the right side. Loading was as follows: Homogenate 0.6% of total, cytosol, microsomes, pure mitochondria, MAM (all 2.4% of total).

Article Snippet: They have been purchased as follows: rabbit anti-Rab29, rabbit anti-Rab32 (Sigma Prestige Antibodies, St. Louis, MO), rabbit anti-Rab38 (abcam, Cambridge UK ab155956, used for IF), mouse anti-Rab38 (Abnova, Taipei, Taiwan, used for Western blot), rabbit anti-PKA RII, rabbit anti-LAMP1 (Santa Cruz Biotechnology, Dallas, TX), mouse anti-ERp57 (StressMarq, Victoria, BC), rabbit anti-Drp1 (Cell Signaling, Danvers, MA), rabbit anti-Syntaxin 7 (ProteinTech, Chicago, IL), mouse and rabbit anti-FLAG (Rockland, Gilbertsville, PA; Sigma, Oakville, ON), goat anti-FACL4, rabbit anti-Rab7, mouse anti-complex 2 (abcam, Cambridge, UK), mouse anti-PDI, (Thermo-Pierce, Rockford, IL), rabbit anti-calnexin antibody (our lab ).

Techniques: Immunofluorescence, Microscopy, Staining, Standard Deviation, Fractionation

Journal: Human reproduction (Oxford, England)

Article Title: The roles of protein disulphide isomerase family A, member 3 (ERp57) and surface thiol/disulphide exchange in human spermatozoa-zona pellucida binding.

doi: 10.1093/humrep/dex007

Figure Lengend Snippet: Figure 2 Expression of ERp57 in human spermatozoa. (A) Western blot analysis of total sperm protein extract probed with anti-ERp57 antibody (1:1000). Lower: Semi-quantitative comparison of ERp57 expression between uncapacitated (UnC) and capacitated (C) sperm- atozoa in western blotting by densitometric analysis. Data represent the mean ± SEM (N = 4). (B) Immuno-localization of ERp57 in human spermatozoa with or without capacitation. Spermatozoa were incu- bated with 1:100 mouse monoclonal anti-ERp57 antibody. They were visualized using Alexa Fluor 488-conjugated goat anti-mouse IgG. Anti- ERp57 antibody pre-absorbed with 10-fold molar excess recombinant ERp57 (Abcam) was used as the control. The results shown are repre- sentative of four replicate experiments examined under a fluorescence microscope with ×600 magnification. (C) Sperm surface expression of ERp57. Human spermatozoa (2 × 106) with or without 0.1 μM calyculin A treatment were resuspended in PBS containing 10 mM of membrane impermeable EZ-Link Sulpho-NHS-SS-Biotin, which labelled the sperm surface proteins. Lower: The biotinylated proteins were purified and resolved on SDS-PAGE followed by western blotting using anti-ERp57 antibody. The results shown are representative of four replicate experi- ments. Upper: Semi-quantitative comparison of ERp57 expression between sperm samples in western blotting by densitometric analysis was shown. Data represent the mean ± SEM (N = 4).

Article Snippet: The role of ERp57 in regulating sperm surface thiol status and sperm functions Uncapacitated spermatozoa were capacitated in EBSS/3% BSA in the presence of rabbit polyclonal anti-ERp57 functional blocking antibody (1:10 000– 1:10; Abcam) at 37°C for 3 h. Anti-ERp57 antibody pre-absorbed with 10-fold molar excess of recombinant ERp57 (Abcam) and isotopic antibody were used as controls.

Techniques: Expressing, Western Blot, Comparison, Recombinant, Control, Microscopy, Membrane, SDS Page

Journal: Human reproduction (Oxford, England)

Article Title: The roles of protein disulphide isomerase family A, member 3 (ERp57) and surface thiol/disulphide exchange in human spermatozoa-zona pellucida binding.

doi: 10.1093/humrep/dex007

Figure Lengend Snippet: Figure 6 Effect of function blocking anti-ERp57 antibody on ZP-binding capacity and sperm surface thiol content of human spermatozoa. (A) Uncapacitated spermatozoa were capacitated in EBSS/3% BSA (control) or in EBSS/3% BSA containing different concentrations of anti-ERp57 antibody with or without recombinant ERp57 preabsorption. The ZP-binding ability of capacitated spermatozoa after treatment was determined by the HZA. The results were expressed as HZI (%). Each point represents the mean ± SEM of the results of 10 HZA using 10 ZP and 10 different spermatozoa sam- ples (N = 10). *P < 0.05 when compared with the corresponding control without treatment. (B) Uncapacitated spermatozoa (Lane 1) were capacitated in EBSS/3% BSA containing 1:100 anti-ERp57 antibody (Lane 2) or isotopic antibody (Lane 3). Cell surface thiol of human spermatozoa (2 × 106/ml) was then labelled with MPB and semi-quantified by western blotting. The results shown are representative of five replicate experiments.

Article Snippet: The role of ERp57 in regulating sperm surface thiol status and sperm functions Uncapacitated spermatozoa were capacitated in EBSS/3% BSA in the presence of rabbit polyclonal anti-ERp57 functional blocking antibody (1:10 000– 1:10; Abcam) at 37°C for 3 h. Anti-ERp57 antibody pre-absorbed with 10-fold molar excess of recombinant ERp57 (Abcam) and isotopic antibody were used as controls.

Techniques: Blocking Assay, Binding Assay, Control, Recombinant, Western Blot

Journal: eLife

Article Title: Co-movement of astral microtubules, organelles and F-actin by dynein and actomyosin forces in frog egg cytoplasm

doi: 10.7554/eLife.60047

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-PDIA3 (Rabbit polyclonal) , Boster Bio , Cat#: PB9772 , IHC (1:1000).

Techniques: Labeling, Recombinant, Expressing, Software

Journal: Heliyon

Article Title: The role of PDIA3 in oral squamous cell carcinoma and its value as A diagnostic and prognostic biomarker

doi: 10.1016/j.heliyon.2023.e22596

Figure Lengend Snippet: PDIA3 expression is up-regulated in OSCC tissues. (A and B) The mRNA expression of PDIA3 in the normal and OSCC tissues in the OSCC dataset from TCGA. (A) Compare all tumor and normal tissues. (B) Only compare paired tumors and normal tissues. (C) The protein expression of PDIA3 by IHC was retrieved from the HPA database. (D) The immunofluorescent staining of PDIA3 in human OSCC tissues and non-tumorous tissues. Left, representative images; right, quantification of the intensity of the PDIA3 staining. Bar = 20 μm. (E) The IHC staining of PDIA3 in human OSCC tissues and normal tissues. Left, representative IHC images. Upper: bar = 200 μm; lower: bar = 50 μm. Right, quantification of the intensity of the PDIA3 staining. (F) PDIA3 mRNA expression in normal tissues and tumors at different T stages. (G) PDIA3 level in normal tissues and OSCC tumors at different N stages. ***, P < 0.001. A P < 0.05 is significantly different.

Article Snippet: The slides were then incubated overnight at 4 °C with primary rabbit polyclonal anti-PDIA3 antibody (1:300; Proteintech, USA).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Heliyon

Article Title: The role of PDIA3 in oral squamous cell carcinoma and its value as A diagnostic and prognostic biomarker

doi: 10.1016/j.heliyon.2023.e22596

Figure Lengend Snippet: The diagnostic and prognostic value of PDIA3 in OSCC. The PDIA3 expression and clinical data were retrieved from the OSCC dataset in TCGA. (A) The ROC curve for analyzing the value of PDIA3 in the diagnosis of OSCC. (B–D) The Kaplan-Meier method was used to analyze the value of PDIA3 in predicting the prognosis of OSCC patients. The Kaplan-Meier curves for (B) OS, (C) DSS and (D) PFI are shown here. The Logrank test was used to compare the curves. A P < 0.05 is significantly different.

Article Snippet: The slides were then incubated overnight at 4 °C with primary rabbit polyclonal anti-PDIA3 antibody (1:300; Proteintech, USA).

Techniques: Diagnostic Assay, Expressing, Biomarker Discovery

Journal: Heliyon

Article Title: The role of PDIA3 in oral squamous cell carcinoma and its value as A diagnostic and prognostic biomarker

doi: 10.1016/j.heliyon.2023.e22596

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of PDIA3 in OSCC.

Article Snippet: The slides were then incubated overnight at 4 °C with primary rabbit polyclonal anti-PDIA3 antibody (1:300; Proteintech, USA).

Techniques:

Journal: Heliyon

Article Title: The role of PDIA3 in oral squamous cell carcinoma and its value as A diagnostic and prognostic biomarker

doi: 10.1016/j.heliyon.2023.e22596

Figure Lengend Snippet: Down-regulation of PDIA3 inhibits the cancerous phenotypes of OSCC cells. The CAL27 and SCC25 cells were transfected with siRNA oligos targeting PDIA3 (designated as siPDIA3). Cells transfected with non-targeting oligos were used as controls (siNC). The untreated cells were also used and designated as ctrl. (A and B) Immunoblotting was performed to measure the protein level of PDIA3 in (A) CAL27 and (B) SCC25 cells. (C and D) The cell viability was measured by CCK8 assay on 24, 48, 72, and 96 h in (C) CAL27 and (D) SCC25 cells transfected with siPDIA3 or non-targeting siRNA. (E and F) The apoptosis of (E) CAL27 and (F) SCC25 cells was measured using an ELISA assay detecting the level of cytosolic nucleosomes. (G and H) The migratory ability of siRNA-transfected (G) CAL27 and (H) SCC25 cells was evaluated using a wound healing assay. Left: representative images; right: quantification of the wound healing rates. Scale bar = 100 μm *, P < 0.05, **, P < 0.01, ***, P < 0.001. A P < 0.05 is significantly different.

Article Snippet: The slides were then incubated overnight at 4 °C with primary rabbit polyclonal anti-PDIA3 antibody (1:300; Proteintech, USA).

Techniques: Transfection, Western Blot, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Wound Healing Assay

Journal: Heliyon

Article Title: The role of PDIA3 in oral squamous cell carcinoma and its value as A diagnostic and prognostic biomarker

doi: 10.1016/j.heliyon.2023.e22596

Figure Lengend Snippet: PDIA3 regulates the PI3K/AKT signaling pathway in OSCC. (A) The mRNA expression of PI3KCD, AKT1-3, and mTOR in OSCC and normal tissues. (B) A heatmap showing the expression levels of PI3KCD, AKT1-3, and mTOR across PDIA3 high and PDIA3 low OSCC tissues. Red: high expression level; blue: low expression level. (C) The correlation between PDIA3 and PI3KCD, AKT1-3, or mTOR was analyzed by Spearman's correlation analysis. (D and E) PDIA3 was silenced by siRNA transfection (siPDIA3). Cells transfected with non-targeting siRNA (siNC) and untreated cells (ctrl) were used as controls. The levels of phosphor-AKT and total AKT were measured by immunoblotting in (D) CAL27 and (E) SCC25 cells. *, P < 0.05, **, P < 0.01, ***, P < 0.001. A P < 0.05 is significantly different.

Article Snippet: The slides were then incubated overnight at 4 °C with primary rabbit polyclonal anti-PDIA3 antibody (1:300; Proteintech, USA).

Techniques: Expressing, Transfection, Western Blot

Journal: Heliyon

Article Title: The role of PDIA3 in oral squamous cell carcinoma and its value as A diagnostic and prognostic biomarker

doi: 10.1016/j.heliyon.2023.e22596

Figure Lengend Snippet: PDIA3 single-gene GSEA functional enrichment analysis. The GSEA for the differential genes related to PDIA3. (A) Enriched functions of the down-regulated genes. (B) Enriched functions of the up-regulated genes. (C) A lollipop plot showing the correlation between PDIA3 expression and immune cell infiltration in OSCC tissues by Spearman analysis. *, P < 0.05, **, P < 0.01, ***, P < 0.001, n.s, not significant. A P < 0.05 is significantly different.

Article Snippet: The slides were then incubated overnight at 4 °C with primary rabbit polyclonal anti-PDIA3 antibody (1:300; Proteintech, USA).

Techniques: Functional Assay, Expressing