glucose regulated protein 94  (Proteintech)

Journal: Experimental & Molecular Medicine

Article Title: Engineered EVs from LncEEF1G - overexpressing MSCs promote fibrotic liver regeneration by upregulating HGF release from hepatic stellate cells

doi: 10.1038/s12276-025-01413-4

Figure Lengend Snippet: a A schematic diagram of the procedures for obtaining MSC-EVs from MSC-conditioned medium. b , c TEM (b) and NTA (c) were used to detect the morphology and size of MSC-EVs. Scale bar, 0.1 μm. d Representative western blotting results showing the expression of specific vesicle-related markers, namely, ALIX, CD63, CD81 and GRP94. e The representative fluorescence images of the uptake of MSC-EVs (fluorescent red) in HSCs were obtained via confocal microscopy. Scale bar, 20 μm.

Article Snippet: After being blocked with nonfat milk (5%) at room temperature for 1 h, the membranes were incubated with primary antibodies (including antibodies against Proliferating Cell Nuclear Antigen (PCNA) (1:1,000; Cell Signaling Technology), HGF (1:1,000; Cell Signaling Technology), TGF-β (1:1,000; Cell Signaling Technology), apoptosis-linked gene-2 interacting protein X (ALIX) (1:1,000; Cell Signaling Technology), CD63 (1:1,000; Abcam), CD81 (1:1,000; Cell Signaling Technology), glucose-regulated protein 94 (GRP94) (1:1,000; Cell Signaling Technology), α-SMA (1:1,000; Abcam), Vimentin (1:1,000; Abcam) and β-actin (1:1,000; Cell Signaling Technology)) overnight at 4 °C, after which they were incubated with secondary antibodies (anti-rabbit IgG, Sigma-Aldrich; anti-mouse IgG, Sigma-Aldrich) for 1 h at room temperature on a table shaker.

Techniques: Western Blot, Expressing, Fluorescence, Confocal Microscopy

Journal: Experimental & Molecular Medicine

Article Title: Engineered EVs from LncEEF1G - overexpressing MSCs promote fibrotic liver regeneration by upregulating HGF release from hepatic stellate cells

doi: 10.1038/s12276-025-01413-4

Figure Lengend Snippet: a TEM and NTA were used to detect the morphology and size of the MSC-EVs and lncEEF1G OE -EVs. Scale bar, 0.1 μm. b Representative western blots of specific vesicle-related markers (ALIX, CD63, CD81 and GRP94). c Representative fluorescence images of the uptake of lncEEF1G OE -EVs (fluorescent red) in HSCs were obtained via confocal microscopy. Scale bar, 20 μm). d RT‒qPCR showing the expression of lncEEF1G in MSC-EVs and lncEEF1G OE -EVs. e A schematic diagram of the application of lncEEF1G OE -EVs to detect whether lncEEF1G OE -EVs stimulate fibrotic liver regeneration. f Serum ALT, AST and LDH levels in different treatment groups ( n = 4 independent biological mouse samples). g The LW/BW ratio in different treatment groups ( n = 4 independent biological mouse samples). h The representative images of H&E-stained samples from different treatment groups. Scale bar, 50 μm). i A quantification of the mitotic frequency determined by counting the numbers of mitotic nuclei and total nuclei in randomly selected fields ( n = 4 independent biological mouse samples). j , k The representative images ( j ) and quantification ( k ) of Ki67 immunohistochemical staining in different treatment groups. Scale bar, 100 μm ( n = 4 independent biological mouse samples). l Western blotting (left) and relative quantification (right) showing the expression of PCNA in liver samples subjected to different treatments ( n = 3 independent experiments). m , n RT‒qPCR ( m ) and representative western blotting ( n , left) results showing the expression of HGF in HSCs isolated from different treatment groups and a quantification ( n , right) of HGF protein levels ( n = 3 independent experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments or four independent biological mouse samples. Statistical significance was determined by a Student’s t -test. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: After being blocked with nonfat milk (5%) at room temperature for 1 h, the membranes were incubated with primary antibodies (including antibodies against Proliferating Cell Nuclear Antigen (PCNA) (1:1,000; Cell Signaling Technology), HGF (1:1,000; Cell Signaling Technology), TGF-β (1:1,000; Cell Signaling Technology), apoptosis-linked gene-2 interacting protein X (ALIX) (1:1,000; Cell Signaling Technology), CD63 (1:1,000; Abcam), CD81 (1:1,000; Cell Signaling Technology), glucose-regulated protein 94 (GRP94) (1:1,000; Cell Signaling Technology), α-SMA (1:1,000; Abcam), Vimentin (1:1,000; Abcam) and β-actin (1:1,000; Cell Signaling Technology)) overnight at 4 °C, after which they were incubated with secondary antibodies (anti-rabbit IgG, Sigma-Aldrich; anti-mouse IgG, Sigma-Aldrich) for 1 h at room temperature on a table shaker.

Techniques: Western Blot, Fluorescence, Confocal Microscopy, Expressing, Staining, Immunohistochemical staining, Quantitative Proteomics, Isolation