Journal:

Article Title: The classical progesterone receptor mediates Xenopus oocyte maturation through a nongenomic mechanism

doi:

Figure Lengend Snippet: Expression of xPR in Xenopus oocytes. (A) Total RNA (1 μg each) from oocytes of the various stages (stages I–III mixed with unknown ratio) were reverse-transcribed followed by PCR amplification using xPR primers encompassing exons E1 and E2. Lane 4 represents a negative control in which PCR was performed directly on input stage VI oocyte RNA without prior reverse transcription (RT). Shown is a representative of four independent experiments with arrows indicating the specifically amplified products. (B) Extracts from stage IV (lane 1), stage V (lane 2), or stage VI (lane 3) oocytes, isolated after collagenase treatment of ovarian tissues, or extracts from collagenase-treated and devitellinated oocytes (lane 4), were immunoblotted with anti-xPR. Equal amounts (50 μg) of proteins were loaded on each lane. The primary antibodies were visualized by incubation with appropriate secondary antibody-horseradish peroxidase conjugates followed by the use of a chemiluminescence kit (ECL, Amersham Pharmacia). Shown is a representative of three independent experiments. (C) Extracts from uninjected (lanes 1, 5, and 9) or Myc-xPR mRNA-injected (lanes 2, 6, and 10) oocytes (each representing half an oocyte), or extracts from untransfected (lanes 3, 7, and 11) or Myc-xPR-transfected (lanes 4, 8, and 12) COS cells (each representing one-tenth of a 3-cm dish) were immunoblotted with the indicated antibodies. Shown is a representative of three independent experiments.

Article Snippet: Antibodies against Xenopus β-integrin (8C8) were purchased from the Developmental Studies Hybridoma Bank at the University of Iowa.

Techniques: Expressing, Reverse Transcription, Amplification, Negative Control, Isolation, Incubation, Injection, Transfection

Journal:

Article Title: The classical progesterone receptor mediates Xenopus oocyte maturation through a nongenomic mechanism

doi:

Figure Lengend Snippet: xPR is a cytosolic protein in Xenopus oocytes. (A) Extracts from intact oocytes (lane 1, equivalent of half an oocyte), isolated nuclei (lane 2, 1 GV), nuclei isolated from oocytes after 15- or 45-min incubation with progesterone (lanes 3 and 4, respectively, 1 GV each), or from enucleated oocytes (lane 5, half an oocyte), were immunoblotted with anti-xPR or anti-Xenopus nucleolin (R2D2) (14). Shown is a representative of five independent experiments. (B) Extracts from intact oocytes (lane 1), 100,000 × g pellet (lane 2), or supernatant (lane 3), each of which were the equivalent of one oocyte, were blotted with anti-xPR or anti-β-integrin. Shown is a representative of five independent experiments.

Article Snippet: Antibodies against Xenopus β-integrin (8C8) were purchased from the Developmental Studies Hybridoma Bank at the University of Iowa.

Techniques: Isolation, Incubation

Journal: Materials Today Bio

Article Title: A biomimetic multimodal nanoplatform combining neutrophil-coated two-dimensional metalloporphyrinic framework nanosheet and exendin-4 to treat obesity-related osteoporosis

doi: 10.1016/j.mtbio.2025.102009

Figure Lengend Snippet: The characterization of DPA@NM@CTZE. (A) Scanning electron microscopy (SEM) image of CTZ. Scale bar: 100 μm. ( B-C ) Transmission electron microscopy (TEM) analysis of CTZ and NM@CTZE. Scale bar: 50 nm, 100 nm, 50 nm. ( D ) Energy-dispersive X-ray spectroscopy (EDS) analysis of CTZ. Scale bar: 2.5 μm. ( E ) X-ray photoelectron spectroscopy (XPS) employed to investigate the surface chemical states of various elements in CTZ. ( F ) X-ray diffraction (XRD) utilized to examine the characteristic diffraction peaks of CTZ. ( G ) Fourier Transform Infrared Spectroscopy (FTIR) was employed to examine the molecular structure of DPA. ( H ) The effectiveness of NM@CTZE synthesis was evaluated through Zeta potential analysis. ( I ) The trend in Zeta potential variation following CTZ loading in Ex-4 was investigated. ( J ) The expression levels of key membrane proteins, including Integrin β1, Integrin β2, and CXCR2, on the surface of NM were analyzed using WB. ( K ) The ROS responsiveness of DPA@NM@CTZE was assessed. Data are presented as Mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3.

Article Snippet: WB using antibodies including: TET2 (Abcam, USA), COL1A1 (Collagen I, Abcam, UK), RUNX2 (Runt-related transcription factor 2, CST, USA), PINK1 (PTEN-induced putative kinase protein 1, Bioss, China), Parkin (E3 ubiquitin-protein ligase parkin, Servicebio, China), LC3B (Light Chain 3, Abmart, China), ATG4B (Autophagy Related 4B Cysteine Peptidase, Proteintech, China), BCL2 (B-cell lymphoma-2, Abmart, China), BECN1 (Beclin-1, Abmart, China), ACTB (Beyotime, China), Integrin β1 (MCE, USA), Integrin β2 (Bio-Techne, USA), CXCR2 (Proteintech, China), HRP-labeled Goat Anti-Rabbit IgG (H + L) (Beyotime, China), HRP-labeled Goat Anti-Mouse IgG (H + L) (Beyotime, China), KEAP1 (Kelch Like ECH Associated Protein 1, Proteintech, China),NRF2 (Nuclear factor erythroid 2-related factor 2, Proteintech, China).

Techniques: Electron Microscopy, Transmission Assay, Spectroscopy, Fourier Transform Infrared Spectroscopy, Zeta Potential Analyzer, Expressing, Membrane

Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 5. Expression changes of key GBM proteins and their response to FN1 Variants in TBMN. Immunofluorescence experiments assessed the expression of Laminin α5β2, COL4A3/4/5 and Integrin β1 in the GBM. The findings revealed non-homogeneous linear structure expression of these key GBM proteins in the presence of FN1 variants, indicating their potential involvement in the pathogenesis of TBMN. HC = healthy control.

Article Snippet: Incubating primary antibodies overnight at 4 ◦C, with specific primary antibodies against HA (1:2000, ab9110, Abcam), Integrin β1 (1:1000, 12,594–1-AP, Proteintech), β-tubulin (1:3000, ab0039, Abways), Laminin α5 (1:1000, ab210957, Abcam), Laminin β2 (1:1000, ab210956, Abcam), Laminin γ1 (1:1000, ab233389, Abcam) in Tris-Buffered Saline Tween-20 (TBST) containing 5 % skim milk.

Techniques: Expressing, Immunofluorescence, Control

Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 6. Abnormal co-localization of FN1 variants with Integrin β1 and its impact on other GBM components. Immunofluorescence studies investigated the rela tionship between FN1 variants and the expression patterns of GBM components. The findings reveal abnormal co-localization of FN1 variants with Integrin β1, accompanied by reduced co-localization of other GBM components. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Incubating primary antibodies overnight at 4 ◦C, with specific primary antibodies against HA (1:2000, ab9110, Abcam), Integrin β1 (1:1000, 12,594–1-AP, Proteintech), β-tubulin (1:3000, ab0039, Abways), Laminin α5 (1:1000, ab210957, Abcam), Laminin β2 (1:1000, ab210956, Abcam), Laminin γ1 (1:1000, ab233389, Abcam) in Tris-Buffered Saline Tween-20 (TBST) containing 5 % skim milk.

Techniques: Immunofluorescence, Expressing

Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 7. Competitive Binding of FN1 Variants to Integrin β1. (A and B) Duolink proximity ligation assay and Co-IP were employed to analyze the protein interaction between FN1 variants and Integrin β1. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Incubating primary antibodies overnight at 4 ◦C, with specific primary antibodies against HA (1:2000, ab9110, Abcam), Integrin β1 (1:1000, 12,594–1-AP, Proteintech), β-tubulin (1:3000, ab0039, Abways), Laminin α5 (1:1000, ab210957, Abcam), Laminin β2 (1:1000, ab210956, Abcam), Laminin γ1 (1:1000, ab233389, Abcam) in Tris-Buffered Saline Tween-20 (TBST) containing 5 % skim milk.

Techniques: Binding Assay, Proximity Ligation Assay, Co-Immunoprecipitation Assay

Journal: Molecular Biology of the Cell

Article Title: GRASP depletion–mediated Golgi destruction decreases cell adhesion and migration via the reduction of α5β1 integrin

doi: 10.1091/mbc.e18-07-0462

Figure Lengend Snippet: Figure 3. GRASP expression rescues the cell migration defects in GRASP knockout cells. (A) GRASP expression rescues the decreased cell migration in GRASP-knockout (KO) HeLa cells. Cells transfected with indicated constructs were tested in a wound-healing assay and images were processed by Wimasis Image Analysis. Images were taken at 0 h and 20 h after scratching. Scale bar, 500 µm. (B) Quantitation of (A). The reduced migration of GRASP55 and GRASP65 KO cells was rescued by expressing GRASP55-GFP and GRASP65-GFP, respectively, but not by GFP alone. Results were presented as mean ± SEM, statistical analysis was performed by comparing to wild type control (ctrl) by student’s t-test. *, p<0.05; **, p<0.01. (C) Western blot of HeLa cells transfected with indicated constructs. α5 integrin heavy chain (α5 integrin HC), GRASP55, GRASP65, GFP and actin were blotted. The reduced protein levels of α5 integrin in GRASP55 and GRASP65 KO cells were rescued by expressing GRASP55-GFP or GRASP65-GFP, respectively, but not by GFP alone.

Article Snippet: After washing, beads were boiled in SDS sample buffer with 100 mM DTT and bound proteins were analyzed by SDS-PAGE, transferred to PVDF membranes and blotted with α5 integrin (Cell Signaling, Cat# 4705), β1 integrin (Cell Signaling, Cat# 4706), Transferrin receptor (Invitrogen, Cat# 13-6800) and β-actin (Proteintech, Cat# 66009-1-Ig) antibodies.

Techniques: Expressing, Migration, Knock-Out, Transfection, Construct, Wound Healing Assay, Quantitation Assay, Control, Western Blot

Journal: Molecular Biology of the Cell

Article Title: GRASP depletion–mediated Golgi destruction decreases cell adhesion and migration via the reduction of α5β1 integrin

doi: 10.1091/mbc.e18-07-0462

Figure Lengend Snippet: Figure 6. GRASP depletion reduces α5 and β1 integrin synthesis but has no effect on their turnover.

Article Snippet: After washing, beads were boiled in SDS sample buffer with 100 mM DTT and bound proteins were analyzed by SDS-PAGE, transferred to PVDF membranes and blotted with α5 integrin (Cell Signaling, Cat# 4705), β1 integrin (Cell Signaling, Cat# 4706), Transferrin receptor (Invitrogen, Cat# 13-6800) and β-actin (Proteintech, Cat# 66009-1-Ig) antibodies.

Techniques:

Journal: PLoS ONE

Article Title: miRNA-29c Suppresses Lung Cancer Cell Adhesion to Extracellular Matrix and Metastasis by Targeting Integrin β1 and Matrix Metalloproteinase2 (MMP2)

doi: 10.1371/journal.pone.0070192

Figure Lengend Snippet: (A) Effects of miR-29c on endogenous integrin β1 protein. Changes in protein expression of Int β1 was determined by Western blotting analysis in 95D 29c , 95D MiNC, 95C 29ci and 95C IhNC. GAPDH was used as an internal loading control. (B) The gray levels of Western blotting are shown by bar gragh. miR-29c mimics inhibit endogenous integrin β1 in 95D cell lines (95D 29c and 95D MiNC), while miR-29c inhibitors promote integrin β1 protein expression. ** p <0.01 (Student's t -test, n = 3). (C) Effects of miR-29c on endogenous MMP2 protein levels by Western blotting. (D) The gray levels of Western blotting are shown by bar gragh. ** p <0.01 (Student's t -test, n = 3). (E) Effects of miR-29c on MMP2 and MMP9 enzyme activity in lung cancer by gelatin zymography. (F) The gray level of every band was measured to check the differences in MMP2 enzyme activity in 95C and 95D cells (The brightest band [95D MiNC] was set to 1 unit to compare with other gray levels). ** p <0.01 (Student's t -test, n = 3). (G) The gray level of every band was measured to check the difference in MMP9 enzyme activity between 95C and 95D cells (The brightest band [95D MiNC] was set to 1 unit to compare with other gray levels). ** p <0.01 (Student's t -test, n = 3).

Article Snippet: The membranes were blocked in 5% non-fat milk in TBST buffer (Tris Buffer Saline containing 0.1% Tween-20) for 1 h at room temperature, and subsequently incubated overnight at 4°C by the appropriately diluted primary antibodies for rabbit monoclonal anti-human integrin β1 and MMP2 (diluted 1∶1000; Cell Signaling Technology).

Techniques: Expressing, Western Blot, Control, Activity Assay, Zymography

Journal: Cancer Medicine

Article Title: The expression and role of SUZ12 in lung adenocarcinoma

doi: 10.1002/cam4.70190

Figure Lengend Snippet: The effect of SUZ12 on metastasis related genes expression was tested by qRT‐PCR and western blotting. sh‐SUZ12 increased MMP1/2/3/9 mRNA expression (A) and TIMP1/2 protein expression (B), while decreased MMP14 mRNA expression (A), MMP1/9/14 (B), TIMP3 (B), and ITGB1/5(F) protein expression, without significantly effected ITGBs mRNA expression (E). oe‐SUZ12 increased MMP14 mRNA (C) and protein (D) expression, while decreased TIMP2 mRNA expression (C) and TIMP1/2 (D) protein expression.

Article Snippet: The list of primary antibodies: SUZ12 (1 μg/mL, Abcam Cambridge, cat no: ab12073), CDK2 (1:1000; Proteintech, USA, cat. no. 10122‐1‐AP), CDK3 (1:2000; Proteintech, USA, cat. no. 55103‐1‐AP), CDK6 (1:2000; Proteintech, USA, cat. no. 14052‐1‐AP), cyclin D1 (1:5000; Proteintech, USA, cat. no. 26939‐1‐AP), cyclin E1 (1:1000; Proteintech, USA, cat. no. 11554‐1‐AP), p18 (1:1000, BOSTER China, cat. no. M03299‐1), p19 (1:1000, BOSTER China, cat. no. MA1075), p53 (1:5000; Proteintech, USA, cat. no. 60283‐2‐Ig), p‐p53 (1:2000; Proteintech, USA, cat. no. 28961‐1‐AP), p57 (1:1000, BOSTER China, cat. no. BM4129), Rb (1:1000, BOSTER China, cat. no. BM4500), pRb (1:1000, BOSTER China, cat. no. BM4338), Bcl‐2 (1:1000; Proteintech, USA, cat. no. 26593‐1‐AP), Bax (1:2000; Proteintech, USA, cat. no. 50599‐2‐lg), E‐cadherin (1:5000; Proteintech, USA, cat. no. 20874‐1‐AP), N‐cadherin (1:3000; Proteintech, USA, cat. no. 22018‐1‐AP), vimentin (1:4000; Proteintech, USA, cat. no. 10366‐1‐AP), MMP1 (1:1000, BOSTER China, cat. no. A00733‐1), MMP2 (1:500, BOSTER China, cat. no. BM4075), MMP9 (1:1000, BOSTER China, cat. no. PB0709), MMP14 (1:1000, BOSTER China, cat. no. BM4119), TIMP1 (1:1000, Bioss China, cat. no. bs‐0415R), TIMP2 (1:1000, Bioss China, cat. no. bs‐10395R), TIMP3 (1:1000; Proteintech, USA, cat. no. 10858‐1‐AP), ITGB1 (1:1000, BOSTER China, cat. no. BM4308), ITGB3 (1:1000, BOSTER China, cat. no. BA1670), ITGB5 (1:1000, BOSTER China, cat. no. A04201‐1), nm23 (1:1000, BOSTER China, cat. no. BA3787), PD‐L1 (1:3000; Proteintech, USA, cat. no. 66248‐1‐Ig), and β‐actin (1:5000; Proteintech, USA, cat. no. 66009‐1‐Ig).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

doi: 10.1038/cddis.2015.50

Figure Lengend Snippet: Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. ( a ) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images ( × 100; scale bars, 200 μ m) are magnified in the right images ( × 600; scale bars, 24 μ m). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~100 cells were counted (** P <0.01, compared with GFP vector). Protein expression was confirmed by western blotting assays using GFP-tagged antibody when equal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous reference protein. ( b and c ) RUFY3 colocalizes with F-actin at the cell periphery. SGC-7901 cells were transiently transfected with pEGFP-C1 or pEGFP-RUFY3. Rhodamine-conjugated phalloidin was used to detect F-actin. After 24 h transfection, cells were fixed and permeabilized. ( b ) Images were captured using a scanning confocal fluorescence microscope and one confocal section is shown in each image. Scale bars, 10 μ m. ( c ) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with F-actin at the cell periphery. The data show mean±S.E.M. (** P <0.01, compared with GFP vector), in which ~40 transfected cells were observed. ( d ) Colocalization of GFP-RIPX and myosinIIb at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of myosinIIb (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 10 μ m. ( e ) Colocalization of GFP-RIPX and integrin β 5 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of integrin β 5 (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 20 μ m. ( f ) Colocalization of GFP-RIPX and integrin α 3 β 1 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. Scale bars, 20 μ m

Article Snippet: The membrane was blocked with 5% nonfat dry milk in TBS-T (20 mM Tris, pH 7.4, 137 mM NaCl, 0.05% Tween-20) for 3 h at room temperature, and the proteins were probed with specific antibodies: GFP and His (GenScript Corporation, Nanjing, China), Flag (Shanghai Kangcheng), PAK1, integrin β 5 and myosinIIb (Cell Signaling), RUFY3 and vinculin (Santa Cruz), integrin α 3 β 1 (Bioss Inc. and Abcam).

Techniques: Over Expression, Transfection, Scratch Wound Assay Assay, Plasmid Preparation, Expressing, Western Blot, Fluorescence, Microscopy, Confocal Microscopy