sheep polyclonal  (R&D Systems)

Journal: Experimental Animals

Article Title: Progranulin deficiency attenuates tubulointerstitial injury in a mouse unilateral ureteral obstruction model

doi: 10.1538/expanim.23-0080

Figure Lengend Snippet: Progranulin (PGRN) and granulin (GRN) expression levels in unilateral ureteral obstruction (UUO)-operated kidneys. (A) mRNA expression levels of Grn in the kidneys of UUO-wild-type (WT) and Control-WT mice at days 3 and 7 after UUO. PGRN mRNA levels in the kidneys were higher in UUO-WT mice than in Control-WT mice. Comparisons among groups were performed using an unpaired t -test. *** P< 0.001. (B) Immunofluorescence staining for PGRN in kidney sections. PGRN expression was localized to the tubules. Blue and green indicate 4′,6-diamidino-2-phenylindole and PGRN, respectively. (C) PGRN and GRN protein levels in UUO-operated kidneys by western blotting. PGRN protein levels were significantly higher in the kidneys of UUO-WT mice on day 3 and 7 after UUO compared to those in Control-WT mice. Comparisons among groups were performed using an unpaired t -test. * P< 0.05. *** P< 0.001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KO, knockout.

Article Snippet: Kidneys were embedded in optimal cutting temperature compound, frozen, cut into 4- μ m sections using a cryostat, fixed in 4% fresh paraformaldehyde for 3 min, and blocked with 10% normal goat serum (Dako, Glostrup, Denmark) for 30 min. Immunohistochemical analyses were then performed using polyclonal sheep anti-PGRN antibodies (1:20 dilution in 1% bovine serum, AF2557; R&D Systems, Minneapolis, MN, USA) overnight at 4°C.

Techniques: Expressing, Control, Immunofluorescence, Staining, Western Blot, Knock-Out

Journal: The Journal of biological chemistry

Article Title: Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43.

doi: 10.1016/j.jbc.2023.105272

Figure Lengend Snippet: Figure 10. The KO of Grn increases the secretion of TDP-43 and LC3-II from MEFs. A, the base sequences of the genome-edited regions of PGRN in WT and KO mice were elucidated by Sanger sequencing. Arrowheads in the WT alignment show the two bases deleted by CRISPR–Cas9 genome editing. B, representative confocal images of PGRN (green) immunostaining of MEFs derived from PGRN WT and KO mice. Nuclei were stained with DAPI (blue). Arrowheads indicate vesicle-like structures. The image surrounded by a white square in WT MEFs was magnified. Scale bar represents 5 μm. C–H, PGRN WT and KO MEFs were cultured for 72 h. The cell, P1, and P2 fractions were prepared as shown in the Experimental procedures section. C, representative immunoblots (PGRN, TDP-43, TSG101, LC3, and α-tubulin) of the cell, P1, and P2 fractions are shown. The dashed square indicates bands of cleaved TDP-43 used for quantification. D, the P1 and P2 fractions treated with or without PNGase F were immunoblotted with PGRN and α-tubulin antibodies. Repre- sentative immunoblots of the cell, P1, and P2 fractions are shown (N = 3). E–H, densitometric data on (E) full-length TDP-43, (F) cleaved TDP-43, (G) TSG101, and (H) LC3-II were calculated from immunoblotting results. Relative signal intensities normalized against PGRN KO MEFs are shown. In bar graphs, data are presented as means ± SD (N = 3). * indicates p < 0.05 by the two-tailed unpaired t test. DAPI, 40,6-diamidino-2-phenylindole; LC3-II, microtubule-associated proteins 1A/1B light chain 3B; MEF, mouse embryonic fibroblast; PGRN, progranulin; TDP-43, transactive response DNA-binding protein 43 kDa; TSG101, tumor susceptibility gene 101 protein.

Article Snippet: * indicates p < 0.05 by the two-tailed un 1A/1B light chain 3B; PGRN, progranulin; TDP-43, transactive response DNA-b Biosciences; a polyclonal sheep antimouse PGRN antibody (catalog no.: AF2557, RRID: AB_2114504) from R&D Systems; a monoclonal mouse anti-WIPI2 antibody (catalog no.: MCA5780GA, RRID: AB_10845951) from Bio-Rad; a biotinconjugated horse antimouse IgG antibody (catalog no.: BA2000, RRID: AB_2313581), biotin-conjugated goat anti-rabbit IgG antibody (catalog no.: BA-1000, RRID: AB_2313606), and biotin-conjugated rabbit antisheep IgG antibody (catalog no.: BA-6000, RRID: AB_2336217) from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG (catalog no.: ab175472, RRID: AB_2636996), and Alexa Fluor 488– conjugated donkey antisheep IgG (catalog no.: ab150177, RRID: AB_2801320) from Abcam.

Techniques: Sequencing, CRISPR, Immunostaining, Derivative Assay, Staining, Cell Culture, Western Blot, Two Tailed Test, Binding Assay

Journal: Scientific Reports

Article Title: Differential organ-specific inflammatory response to progranulin in high-fat diet-fed mice

doi: 10.1038/s41598-020-80940-8

Figure Lengend Snippet: General and biochemical parameters of experimental groups after 12-weeks of HFD.

Article Snippet: Immunohistochemical analyses were performed using the following commercially available antibodies: polyclonal sheep anti-PGRN antibody (R&D Systems), monoclonal mouse anti-megalin antibody (Santa Cruz Biotechnology, CA, USA), and polyclonal rat anti-LAMP1 antibody (Abcam, Cambridge, UK).

Techniques:

Journal: Scientific Reports

Article Title: Differential organ-specific inflammatory response to progranulin in high-fat diet-fed mice

doi: 10.1038/s41598-020-80940-8

Figure Lengend Snippet: Grn mRNA and protein expression levels in the kidney and adipose tissue of the WT-SD and WT-HFD groups. ( A ) Immunofluorescence staining for PGRN shows PGRN expression primarily localized to the tubules. ( B ) Grn mRNA expression levels in the kidney and adipose tissue by real-time PCR. There is no difference in the renal Grn mRNA expression level between the WT-SD and WT-HFD groups. Conversely, the adipose tissue Grn mRNA expression level is significantly higher in the WT-HFD group than in the WT-SD group. Student’s unpaired t test.

Article Snippet: Immunohistochemical analyses were performed using the following commercially available antibodies: polyclonal sheep anti-PGRN antibody (R&D Systems), monoclonal mouse anti-megalin antibody (Santa Cruz Biotechnology, CA, USA), and polyclonal rat anti-LAMP1 antibody (Abcam, Cambridge, UK).

Techniques: Expressing, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Differential organ-specific inflammatory response to progranulin in high-fat diet-fed mice

doi: 10.1038/s41598-020-80940-8

Figure Lengend Snippet: Kidney and adipose tissue mRNA expression levels of inflammatory cytokines in each group. ( A ) Kidney. ( B ) Adipose tissue. Two-way ANOVA was used to evaluate the individual effects of the presence/absence of PGRN and diet type and their interactions. In the kidney, Ccl2 and Vcam1 mRNA expression levels are significantly associated with both PGRN status and diet type. In contrast to the effect on kidney mRNA expression levels, PGRN status and diet type enhance adipose tissue Ccl2 mRNA expression levels in the WT-HFD group. For the mRNA expression levels of Vcam1 in the kidney and Ccl2 in the adipose tissue, main effects were confirmed for PGRN status and diet type and for an interaction between PGRN status and diet type based on post hoc Tukey’s honestly significant difference test. Data are shown as fold changes compared to the WT-SD group, * p < 0.05 versus WT-HFD, † p < 0.05 versus KO-SD.

Article Snippet: Immunohistochemical analyses were performed using the following commercially available antibodies: polyclonal sheep anti-PGRN antibody (R&D Systems), monoclonal mouse anti-megalin antibody (Santa Cruz Biotechnology, CA, USA), and polyclonal rat anti-LAMP1 antibody (Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Differential organ-specific inflammatory response to progranulin in high-fat diet-fed mice

doi: 10.1038/s41598-020-80940-8

Figure Lengend Snippet: Protein expression levels of megalin in each group. ( A ) Immunohistochemistry for megalin (400 × , scale bar: 20 µm). Staining is primarily localized to the proximal tubules. ( B ) Staining intensity of megalin protein in the mouse kidney is presented as fold changes compared to the WT-SD group. Two-way ANOVA shows a significant main effect of only PGRN status on megalin protein expression. p < 0.01 for PGRN status; p = 0.64 for diet type.

Article Snippet: Immunohistochemical analyses were performed using the following commercially available antibodies: polyclonal sheep anti-PGRN antibody (R&D Systems), monoclonal mouse anti-megalin antibody (Santa Cruz Biotechnology, CA, USA), and polyclonal rat anti-LAMP1 antibody (Abcam, Cambridge, UK).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: Differential organ-specific inflammatory response to progranulin in high-fat diet-fed mice

doi: 10.1038/s41598-020-80940-8

Figure Lengend Snippet: mRNA expression levels of Lrp2 and Grn in cultured mouse PTECs stimulated with TNFα. ( A ) The mRNA expression levels of Lrp2 and Grn are reduced in a dose-dependent manner in PTECs stimulated with TNFα. Tukey’s honestly significant difference test. * p < 0.05 versus no TNFα stimulation. ( B ) Effects of PGRN siRNA on megalin expression in PTECs stimulated with 10 ng/mL TNFα. Two-way ANOVA shows significant main effects of PGRN knockdown and TNFα stimulation on the expression of megalin ( p < 0.01 and p = 0.04, respectively), without significant interaction ( p = 0.64), showing that knocking down PGRN reduces megalin expression.

Article Snippet: Immunohistochemical analyses were performed using the following commercially available antibodies: polyclonal sheep anti-PGRN antibody (R&D Systems), monoclonal mouse anti-megalin antibody (Santa Cruz Biotechnology, CA, USA), and polyclonal rat anti-LAMP1 antibody (Abcam, Cambridge, UK).

Techniques: Expressing, Cell Culture, Knockdown