Journal: Iranian Journal of Microbiology

Article Title: Yeast-mediated display: probing Helicobacter pylori HopQ and CEACAM1 interaction

doi: 10.18502/ijm.v17i6.20366

Figure Lengend Snippet: SDS-PAGE and Western blot analyses. In panel A, the left image shows Coomassie Blue staining of S. cerevisiae expressing Aga2-C1ND (lanes 1 and 2). The middle blot shows two distinct bands at 50 kDa and 24 kDa detected using an HRP-conjugated anti-GFP antibody. Lane 1 corresponds to S. cerevisiae expressing the GFP-C1ND fusion construct. The presence of a 27 kDa band suggests cleavage of EGFP from its fusion partner. The right image shows signals detected by the anti-C1ND antibody for non-EGFP-tagged C1ND. The 24 kDa band corresponds to the Aga2-C1ND fusion protein. In panel B, the left image shows positive signals detected using the anti-C1ND antibody. Lane 1 shows the expression of the GFP-C1ND fusion protein, with bands at 24 kDa and 50 kDa. Lanes 2 and 3 represent strains expressing the Aga2-C1ND fusion protein, showing a band at 24 kDa. The positive control (lane 4) expresses the intact C1ND fragment, displaying a band at 12 kDa. The right image shows detection of the EGFP-C1ND fusion protein (50 kDa, lane 1) in a positive transformant using an HRP-conjugated anti-His-tag antibody. The positive control in this blot (lane 2) corresponds to an irrelevant His-tagged protein. In all blots, “C−“ indicates non-transformed lysate. Blots in panels A and B were developed using ECL and DAB staining, respectively.

Article Snippet: Immunoblots were probed overnight at 4°C with HRP-labeled antibodies against GFP (1:2000; Acris, USA), His-tag (1:1000; Sigma, USA), and C1ND (1:3000; Sigma, USA).

Techniques: SDS Page, Western Blot, Staining, Expressing, Construct, Positive Control, Transformation Assay

Journal: Iranian Journal of Microbiology

Article Title: Yeast-mediated display: probing Helicobacter pylori HopQ and CEACAM1 interaction

doi: 10.18502/ijm.v17i6.20366

Figure Lengend Snippet: Confocal microscopy images showing the surface expression of the Aga2-EGFP-G4S-C1ND fusion protein in S. cerevisiae , indicated by arrows. (A) Green fluorescence from the EGFP tag. (B) Red surface fluorescence of recombinant yeast cells stained with PE-conjugated anti-GFP antibody. (C) Merged image of panels A and B. (D) Brightfield image. (E) No fluorescence in the negative control. Scale bar: 5 μm

Article Snippet: Immunoblots were probed overnight at 4°C with HRP-labeled antibodies against GFP (1:2000; Acris, USA), His-tag (1:1000; Sigma, USA), and C1ND (1:3000; Sigma, USA).

Techniques: Confocal Microscopy, Expressing, Fluorescence, Recombinant, Staining, Negative Control

Journal: Thrombosis Journal

Article Title: Paxillin is an intrinsic negative regulator of platelet activation in mice

doi: 10.1186/1477-9560-12-1

Figure Lengend Snippet: Pxn-KD platelets exhibit augmented outside-in signaling. (A) Control and Pxn-KD platelets treated with 50 ng/mL convulxin were allowed to adhere on immobilized fibrinogen for 30 min. The platelets were then fixed and stained with an anti-GFP antibody (green; left panel) and rhodamine-conjugated phalloidin (red; middle panel). The merged images show colocalization of GFP and actin staining (yellow; right panel). Original magnification, ×600; Bar, 5 μm. Data are representative of three independent experiments. (B) Platelets treated with or without 50 ng/mL convulxin were incubated in dishes coated with 400 μg/mL fibrinogen for 30 min. The area of cell spreading was quantified by ImageJ software. The mean platelet size on BSA was subtracted from the total spread area on fibrinogen to determine the actual increase in platelet spreading. The horizontal bar denotes the mean, and each symbol denotes an individual cell ( n = 281–394 cells). (C) Clot retraction of platelet-rich plasma consisting of diluted human plasma and control or Pxn-KD platelets was initiated by 0.1 U/mL thrombin and then photographed at 0, 45, 90, and 120 min. (D) Clot retraction was quantified by measuring serum formation extruded by clot retraction. Columns and error bars represent the mean ± s.d. ( n = 3). Open bars: control platelets; black bars: Pxn-KD platelets. Statistical significance was determined using Student’s t -test. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. control.

Article Snippet: The following antibodies and agonists were obtained from the specified suppliers: PAC-1 monoclonal antibody (mAb), anti-mouse P-selectin mAb (RB40.34), anti-paxillin mAb (clone 349), and anti-Hic-5 mAb (BD Biosciences, San Jose, CA); horseradish peroxidase-conjugated anti-green fluorescent protein (GFP) polyclonal antibody (Acris Antibodies, Himmelreich, Germany); phycoerythrin (PE)-Cy7-conjugated anti-mouse IgM (eBioscience, San Diego, CA); anti-talin mAb (clone 8D4); anti-phosphotyrosine mAb (clone 4G10), and BAPTA-AM (Millipore, Billerica MA); human fibrinogen and epinephrine (Sigma-Aldrich, St. Louis, MO); anti-vinculin mAb (V284) (Chemicon, Billerica, MA); anti-mouse GPVI mAb (Six.E10), anti-mouse GPIbα mAb (Xia.G5), and anti-mouse integrin αIIbβ3 mAb (Leo.D2 and clone JON/A) (Emfret Analytics, Eibelstadt, Germany); anti-α-actin mAb (D6F6), anti-FAK polyclonal antibody, and anti-Src mAb (32G6) (Cell Signaling Technology, Danvers, MA); anti-Rap1b polyclonal antibody and anti-protein kinase Cα mAb (M4) (Upstate Cell Signaling Solutions, Lake Placid, NY); allophycocyanin (APC)-conjugated anti-rat IgG polyclonal antibody (R& D Systems, Minneapolis, MN); convulxin (ALEXIS Biochemicals, Plymouth Meeting, PA); AYPGKF (Invitrogen, Carlsbad, CA); ADP (MC medical, Tokyo, Japan); U46619 (Cayman Chemical, Ann Arbor, MI).

Techniques: Control, Staining, Incubation, Software, Clinical Proteomics

Journal: Nature Communications

Article Title: Radial glia integrin avb8 regulates cell autonomous microglial TGFβ1 signaling that is necessary for microglial identity

doi: 10.1038/s41467-025-57684-y

Figure Lengend Snippet: Mouse Line and Reagent List

Article Snippet: Antibody , EGFP , Origene , Goat polyclonal antibody. Cat#: R1091P. RRID:AB_1002036. , Used at 1:2000..

Techniques: RNAscope, Multiplex Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-7 inhibits progression of glioblastoma by impairing autophagy resolution, energy metabolism and ECM remodeling

doi: 10.1186/s13046-025-03504-6

Figure Lengend Snippet: MiR-7 significantly inhibits the autophagic flux. A-B . Representative Western Blots of autophagy markers p62 and LC3BI/II and quantification of autophagy flux in SH-SY5Y ( A ) and U87-MG ( B ) cell lines transfected with control mimics (CM) miR-7-5p mimics (miR-7). Cells were treated with 200nM of CQ or vehicle for 6 h. HSP90 was used as loading control. Right panel shows the quantification of LC3BII/I ratio normalized by HSP90 (scaled to 1). Data represents means ± SEM. * P <0.05, significantly different from CM cells. ns, not significantly different. C . Measurement of autophagy flux in CM and miR-7 overexpressing U87-MG cells co-transfected with the mCherry-GFP-LC3 tandem reporter. Merged images of two representative cells per condition and quantification of the average number of autolysophagosome (Cherry + and GFP - puncta) per cell, ( n =3 cells per field, 6 fields per experiment). Data represent the mean of three individual experiments ± SEM. ** P <0.01 compared with CM (normalized to 1). Scale bar: 5 μm

Article Snippet: Other antibodies used are SNAP29 (ProteinTech, 12704-1-AP), GFP (Origene, TA1500), and HSP90 (BD Bioscience, 610419).

Techniques: Western Blot, Transfection, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-7 inhibits progression of glioblastoma by impairing autophagy resolution, energy metabolism and ECM remodeling

doi: 10.1186/s13046-025-03504-6

Figure Lengend Snippet: MiR-7 posttranscriptionally regulates the expression of the SNARE complex proteins STX17 and SNAP29. A . qRT-PCR analysis of hSTX17 and hSNAP29 mRNA expression levels in U87-MG cells overexpressing control mimic (CM) or miR-7-5p (miR-7). B . Representative Western blots of hSTX17 and hSNAP2 in cells treated as in A. HSP90 was used as a loading control. Right graph shows relative protein expression normalized to HSP90. Data correspond to the means ± SEM of three experiments performed in triplicate. *** P < 0.001, significantly different from cells transfected with CM (normalized to 1). C . Luciferase reporter activity of STX17 3’UTR in HEK293 cells transfected with CM or miR-7 and with 3’UTRs with wild-type (WT) or the constructs containing the indicated point mutations (PM) or double point mutations (DPM) (upper panels, highlighted in red). Data are expressed as relative luciferase activity compared with the activity in control samples cotransfected with an equal concentration of CM and correspond to the means ± SEM of three experiments performed in triplicate. ** P < 0.01, significantly different from cells cotransfected with CM and the WT or PM or DPM vectors of the 3′UTRs. D . Luciferase reporter activity of SNAP29 3’UTR as indicated in C. * P < 0.05, *** P < 0.001. E-F . Rescue of autophagy flux blockade induced by miR-7. Representative Western blot of autophagy markers p62 and LC3BI/II in cell transfected with CM or miR-7 and a control GFP plasmid or a plasmid expressing STX17 ( E , STX17-GFP) and SNAP29 ( F , SNAP29-GFP) ORF regions lacking their 3’UTR. Cells were treated with 200nM of CQ for 6 h. HSP90 was used as loading control. ( E , F , right panels). Immunofluorescence confocal imaging cells transfected the STX17-GFP ( E ) and SNAP29-GFP ( F ) plasmids showing colocalization with endogenous STX17 or SNAP29 (red). Dapi (blue) was used to stain the nucleus. Scale bar: 10 μm

Article Snippet: Other antibodies used are SNAP29 (ProteinTech, 12704-1-AP), GFP (Origene, TA1500), and HSP90 (BD Bioscience, 610419).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Transfection, Luciferase, Activity Assay, Construct, Concentration Assay, Plasmid Preparation, Immunofluorescence, Imaging, Staining