Journal: Nutrients

Article Title: Beneficial Effects of Isoflavones in the Kidney of Obese Rats Are Mediated by PPAR-Gamma Expression

doi: 10.3390/nu12061624

Figure Lengend Snippet: Evaluation of renal function. Rats in the CTL group, the DH group, and the DH and ISO group at baseline, 60, 90, and/or 120 days. ( A ) Creatinine clearance, ( B ) plasma urea, ( C ) plasma creatinine, ( D ) uric acid, ( E ) fractional sodium excretion, and ( F ) urinary protein. ( G ) Light microscopy of kidney injury molecule-1 (KIM-1) and quantitative analyses of kidney sections stained for KIM-1. Data are reported as mean ± standard error. The significance level for a null hypothesis was set at 5% ( p < 0.05). (*) compared to the CTL group at 60 days, (**) compared to the DH group at 60 days, (¥) compared to the CTL group at 90 days, (¥¥) compared to the DH group at 90 days, (₸) compared to the CTL group at 120 days, and (₸₸) compared to the DH group at 120 days.

Article Snippet: Paraffin sections were subjected to alcohol and xylene gradient solutions, antigen retrieval, protein block, and incubation with primary antibodies against CD31 (1:200, mouse clone JC70a endothelial, M082, Dako, CA, USA), CD68 (1:200, mouse monoclonal clone, M0876, Dako, CA, USA), and kidney injury molecule 1 (KIM-1) (1:200, rabbit IgG, H07H, Sino Biologica, BJ, CH) overnight at 4 °C.

Techniques: Clinical Proteomics, Light Microscopy, Staining

Journal: Nature Communications

Article Title: Neuraminidase 1 promotes renal fibrosis development in male mice

doi: 10.1038/s41467-023-37450-8

Figure Lengend Snippet: a Relative NEU1, NEU2, NEU3, and NEU4 mRNA levels. Data analysis from Nephroseq database (‘Ju CKD Tublnt’ dataset, median-centered log2). n = 31 samples in control group, n = 123 samples in CKD group. Unpaired t -test. ns, no significant difference. nd, not detected. b The expression of NEU1 (median-centered log2) in kidney specimens from control ( n = 21 samples) and patients with chronic kidney disease (CKD, n = 149 samples). Unpaired t -test. Data analysis from Nephroseq database (‘Ju CKD Glom’ dataset). c The mRNA levels of NEU1 in kidney specimens of CKD ( n = 53 samples) and control ( n = 8 samples) in GSE66494 dataset (Probe ID: A_24_P394533). Unpaired t -test. d Tissue adjacent sections of kidney from patients with non-renal fibrosis or renal fibrosis by immunohistochemistry, Masson staining, and HE staining. Scale bar = 20 μm. NRF non-renal fibrosis, RF Renal fibrosis. n = 8 samples per group. e – g Quantification of NEU1 expression ( e ), fibrotic area ( f ), and score of kidney damage ( g ) based on immunohistochemistry, Masson, or HE staining in ( d ). Data were presented as mean ± SD. n = 8 samples per group. Unpaired two-tailed t -test. IOD: integrated optical density. h The correlation of NEU1 expression and degree of tubular degeneration ( n = 16, Pearson χ 2 test). i – k Pearson’s correlation of NEU1 with serum creatinine level ( i ), blood urea nitrogen (BUN) ( j ), and glomerular filtration rate (GFR) ( k ) ( n = 16, Pearson χ 2 test). l – n Representative images of co-immunofluorescence staining of NEU1 and KIM1 in kidney tissues of non-renal fibrosis and renal fibrotic patients ( l ). Fluorescence intensity of NEU1 and KIM1 in diagram k-up ( m ) and k-down ( n ), Image J software was used for statistics. Scale bars = 20 μm. n = 3 samples per group. a – c Data are presented as box-and-whisker plots, solid line inside box indicates the median, the bottom and top of box represent first and third quartiles, and the bottom and top whisker show the minimum and maximum, respectively. All tests were two-tailed.

Article Snippet: The primary antibodies used were as follows: anti-NEU1 (Proteintech, 67032-1-Ig, 1:1000 for IB, 1:100 for PLA), anti-NEU1(Santa Cruz, sc-166824, 1:100 for IP, 1:100 for IF, and 1:100 for IHC), anti-E-cadherin (CST, 3195s, 1:1000 for IB, 1:400 for IF, and 1:400 for ICH), anti-Vimentin (CST, 5741s, 1:1000 for IB, 1:400 for IF, 1:400 for ICH), anti-Snail (CST, 3879s, 1:1000 for IB and 1:400 for ICH), anti-Slug (CST, 9585s, 1:1000 for IB and 1:400 for ICH), anti-GAPDH (CST, 5174s, 1:1000 for IB), anti-α-smooth muscle actin (CST, 19245s, 1:1000 for IB and 1:400 for ICH), anti-HA-tag (Santa, sc-7392, 1:1000 for IB and 1:100 for IP), anti-Flag-tag (Affinity, T0053, 1:1000 for IB and 1:100 for IP), anti-ALK5 (Affinity, AF5347, 1:1000 for IB, 1:300 for IF, and 1:100 for IP, 1:100 for PLA), anti-p-ALK5 (Affinity, AF8080, 1:1000 for IB and 1:100 for IHC), anti-CD68 (CST, 97778, 1:200 for IHC), anti-p-NFκB (CST, 3037S, 1:200 for IHC), anti-Na/K ATPase (Abcam, ab254025, 1:200 for IF), anti-KIM1 (Cloud-Clone Corp, LAA785Mu81, 1:100 for IF), anti-Smad2 (CST, #5339, 1:1000 for IB), anti-Smad3 (CST, #9523, 1:1000 for IB), anti-p-Smad2 (CST, #3108, 1:1000 for IB), anti-p-Smad3 (CST, #9520, 1:1000 for IB), anti-LAMP1 (Abcam, ab24170, 1:200 for IHC), anti-PPCA (Abcam, ab184553, 1:1000 for IB), anti-biotinylated SNL lectin (Vector Labs, B-1305-2, 1:500 for IB).

Techniques: Expressing, Immunohistochemistry, Staining, Two Tailed Test, Filtration, Immunofluorescence, Fluorescence, Software, Whisker Assay

Journal: Nature Communications

Article Title: Neuraminidase 1 promotes renal fibrosis development in male mice

doi: 10.1038/s41467-023-37450-8

Figure Lengend Snippet: a Scheme of the experimental approach. UUO, unilateral ureteral obstruction. b The gross appearance of kidneys (Scale bar, 1 mm), Hematoxylin and eosin (HE, Scale bar, 20 μm), Masson’s trichrome staining (Scale bar, 20 μm), immunohistochemistry staining of CD68, and p-NFκB (Scale bar, 50 μm) from control (Ctrl) and Neu1 CKO mice 10 days after UUO. The red arrow indicates positive cells. n = 3 mice per group. c The ratio of left renal weight to tibia length (TL). n = 12 mice per group. ( d ) Statistical results for interstitial collagen analyzed by Image Pro-Plus software. n = 3 mice per group. e , f Morphometric analysis, assessing percentage of tubular necrosis index ( e ) and tubulointerstitial inflammation index ( f ). n = 3 mice per group. g , h Quantitative results of CD68 ( g ) and p-NFκB ( h ). n = 3 mice per group. i Kim1 mRNA levels. n = 4 mice per group. j Images of immunofluorescence staining. Scale bars, 20 μm. k Statistical analysis of staining double-positive cells of E-cadherin and Vimentin. HPF, high power field. n = 3 mice per group. l , m EMT and extracellular matrix-associated gene mRNA levels. n = 3 mice per group. Gene expression levels were normalized to Gapdh . All statistic data were presented as mean ± SD, two-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: The primary antibodies used were as follows: anti-NEU1 (Proteintech, 67032-1-Ig, 1:1000 for IB, 1:100 for PLA), anti-NEU1(Santa Cruz, sc-166824, 1:100 for IP, 1:100 for IF, and 1:100 for IHC), anti-E-cadherin (CST, 3195s, 1:1000 for IB, 1:400 for IF, and 1:400 for ICH), anti-Vimentin (CST, 5741s, 1:1000 for IB, 1:400 for IF, 1:400 for ICH), anti-Snail (CST, 3879s, 1:1000 for IB and 1:400 for ICH), anti-Slug (CST, 9585s, 1:1000 for IB and 1:400 for ICH), anti-GAPDH (CST, 5174s, 1:1000 for IB), anti-α-smooth muscle actin (CST, 19245s, 1:1000 for IB and 1:400 for ICH), anti-HA-tag (Santa, sc-7392, 1:1000 for IB and 1:100 for IP), anti-Flag-tag (Affinity, T0053, 1:1000 for IB and 1:100 for IP), anti-ALK5 (Affinity, AF5347, 1:1000 for IB, 1:300 for IF, and 1:100 for IP, 1:100 for PLA), anti-p-ALK5 (Affinity, AF8080, 1:1000 for IB and 1:100 for IHC), anti-CD68 (CST, 97778, 1:200 for IHC), anti-p-NFκB (CST, 3037S, 1:200 for IHC), anti-Na/K ATPase (Abcam, ab254025, 1:200 for IF), anti-KIM1 (Cloud-Clone Corp, LAA785Mu81, 1:100 for IF), anti-Smad2 (CST, #5339, 1:1000 for IB), anti-Smad3 (CST, #9523, 1:1000 for IB), anti-p-Smad2 (CST, #3108, 1:1000 for IB), anti-p-Smad3 (CST, #9520, 1:1000 for IB), anti-LAMP1 (Abcam, ab24170, 1:200 for IHC), anti-PPCA (Abcam, ab184553, 1:1000 for IB), anti-biotinylated SNL lectin (Vector Labs, B-1305-2, 1:500 for IB).

Techniques: Staining, Immunohistochemistry, Software, Immunofluorescence, Expressing

Journal: Nature Communications

Article Title: Neuraminidase 1 promotes renal fibrosis development in male mice

doi: 10.1038/s41467-023-37450-8

Figure Lengend Snippet: a Scheme of the experimental approach. The mice were intraperitoneally injected with folic acid (250 mg/kg). b The gross appearance of whole kidneys from control (Ctrl) and Neu1 CKO mice 4 weeks after folic acid injection. Scale bar, 1 mm. c The ratio of left renal weight to body weight (BW). n = 6 mice per group. d , e Creatinine ( d ) and blood urea nitrogen ( e ) in serum measured by ELISA. n = 6 mice per group. f Kim1 mRNA levels. n = 4 mice per group. g Hematoxylin and eosin (HE), Masson’s trichrome staining, and immunohistochemistry staining of CD68 and p-NFκB in kidney sections from Ctrl and Neu1 CKO mice 28 days after folic acid administration. n = 3 mice per group. Scale bar, 50 μm. The red arrow indicates positive cells. h – k Quantification of tubular injury score ( h ), fibrosis area ( i ), staining of CD68 ( j ), and p-NFκB ( k ). n = 3 mice per group. l , m EMT and extracellular matrix associate gene mRNA level. n = 4 mice per group. Gene expression levels were normalized to Gapdh . All statistic data were presented as mean ± SD. Two-way ANOVA followed by Tukey’s multiple comparisons test. All tests were two-tailed.

Article Snippet: The primary antibodies used were as follows: anti-NEU1 (Proteintech, 67032-1-Ig, 1:1000 for IB, 1:100 for PLA), anti-NEU1(Santa Cruz, sc-166824, 1:100 for IP, 1:100 for IF, and 1:100 for IHC), anti-E-cadherin (CST, 3195s, 1:1000 for IB, 1:400 for IF, and 1:400 for ICH), anti-Vimentin (CST, 5741s, 1:1000 for IB, 1:400 for IF, 1:400 for ICH), anti-Snail (CST, 3879s, 1:1000 for IB and 1:400 for ICH), anti-Slug (CST, 9585s, 1:1000 for IB and 1:400 for ICH), anti-GAPDH (CST, 5174s, 1:1000 for IB), anti-α-smooth muscle actin (CST, 19245s, 1:1000 for IB and 1:400 for ICH), anti-HA-tag (Santa, sc-7392, 1:1000 for IB and 1:100 for IP), anti-Flag-tag (Affinity, T0053, 1:1000 for IB and 1:100 for IP), anti-ALK5 (Affinity, AF5347, 1:1000 for IB, 1:300 for IF, and 1:100 for IP, 1:100 for PLA), anti-p-ALK5 (Affinity, AF8080, 1:1000 for IB and 1:100 for IHC), anti-CD68 (CST, 97778, 1:200 for IHC), anti-p-NFκB (CST, 3037S, 1:200 for IHC), anti-Na/K ATPase (Abcam, ab254025, 1:200 for IF), anti-KIM1 (Cloud-Clone Corp, LAA785Mu81, 1:100 for IF), anti-Smad2 (CST, #5339, 1:1000 for IB), anti-Smad3 (CST, #9523, 1:1000 for IB), anti-p-Smad2 (CST, #3108, 1:1000 for IB), anti-p-Smad3 (CST, #9520, 1:1000 for IB), anti-LAMP1 (Abcam, ab24170, 1:200 for IHC), anti-PPCA (Abcam, ab184553, 1:1000 for IB), anti-biotinylated SNL lectin (Vector Labs, B-1305-2, 1:500 for IB).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Neuraminidase 1 promotes renal fibrosis development in male mice

doi: 10.1038/s41467-023-37450-8

Figure Lengend Snippet: a Schematic diagram of NEU1 overexpression in mice. Mice were in situ injected with AAV9 encoding NEU1 or scramble. After the injection for 5 weeks, the mice were subjected to UUO surgery. b mRNA levels of Neu1 in the cortices of kidneys. n = 3 mice per group. c NEU1 protein levels in the kidneys of AAV9-Ctrl and AAV9-NEU1 mice. n = 2 mice per group. d The gross appearance of kidneys (Scale bar, 1 mm), kidney cross-sections stained with HE (Scale bar, 50 μm), and Masson’s trichrome (Scale bar, 100 μm). n = 6 mice per group. e The ratio of left renal weight to body weight (BW, mg/g). AAV9-Ctrl, n = 11; AAV9-NEU1, n = 10. f Statistical analysis of tubular injury score. n = 6 mice per group. g Statistical results for interstitial collagen in d analyzed by Image Pro-Plus software. n = 6 mice per group. h Kim1 mRNA level. n = 3 mice per group. i mRNA levels of the indicated genes in kidneys. n = 3 mice per group. Gene expression levels were normalized to Gapdh . j – n Images of immunohistochemical staining using indicated antibodies. Scale bars, 20 μm. n = 6 mice per group. o mRNA levels of the indicated genes in kidneys. n = 3 mice per group. All statistic data were presented as mean ± SD. Unpaired two-tailed t -test.

Article Snippet: The primary antibodies used were as follows: anti-NEU1 (Proteintech, 67032-1-Ig, 1:1000 for IB, 1:100 for PLA), anti-NEU1(Santa Cruz, sc-166824, 1:100 for IP, 1:100 for IF, and 1:100 for IHC), anti-E-cadherin (CST, 3195s, 1:1000 for IB, 1:400 for IF, and 1:400 for ICH), anti-Vimentin (CST, 5741s, 1:1000 for IB, 1:400 for IF, 1:400 for ICH), anti-Snail (CST, 3879s, 1:1000 for IB and 1:400 for ICH), anti-Slug (CST, 9585s, 1:1000 for IB and 1:400 for ICH), anti-GAPDH (CST, 5174s, 1:1000 for IB), anti-α-smooth muscle actin (CST, 19245s, 1:1000 for IB and 1:400 for ICH), anti-HA-tag (Santa, sc-7392, 1:1000 for IB and 1:100 for IP), anti-Flag-tag (Affinity, T0053, 1:1000 for IB and 1:100 for IP), anti-ALK5 (Affinity, AF5347, 1:1000 for IB, 1:300 for IF, and 1:100 for IP, 1:100 for PLA), anti-p-ALK5 (Affinity, AF8080, 1:1000 for IB and 1:100 for IHC), anti-CD68 (CST, 97778, 1:200 for IHC), anti-p-NFκB (CST, 3037S, 1:200 for IHC), anti-Na/K ATPase (Abcam, ab254025, 1:200 for IF), anti-KIM1 (Cloud-Clone Corp, LAA785Mu81, 1:100 for IF), anti-Smad2 (CST, #5339, 1:1000 for IB), anti-Smad3 (CST, #9523, 1:1000 for IB), anti-p-Smad2 (CST, #3108, 1:1000 for IB), anti-p-Smad3 (CST, #9520, 1:1000 for IB), anti-LAMP1 (Abcam, ab24170, 1:200 for IHC), anti-PPCA (Abcam, ab184553, 1:1000 for IB), anti-biotinylated SNL lectin (Vector Labs, B-1305-2, 1:500 for IB).

Techniques: Over Expression, In Situ, Injection, Staining, Software, Expressing, Immunohistochemical staining, Two Tailed Test

Journal: Nature Communications

Article Title: Neuraminidase 1 promotes renal fibrosis development in male mice

doi: 10.1038/s41467-023-37450-8

Figure Lengend Snippet: a , b HK-2 cells transfected with siNEU1 ( a ) or NEU1 full-length plasmid ( b ) for 24 h and treated with TGFβ (10 ng/ml) for 24 h. Then the cells were incubated with cycloheximide (CHX, 20 μg/ml) for the indicated periods of time (0, 2, 4, 8, 12, 24 h) (left). Lysates were harvested from the cells and analyzed by western blots. Quantitation of ALK5 protein levels were shown in the right pane. n = 3 samples per group. Unpaired t -test. c – f Western blots ( c , e ) and quantitative results ( d , f ) of p-ALK5, ALK5, p-SMAD2/3, and SMAD2/3 in HK-2 cells transfected with siNEU1 or NEU1 plasmid for 24 h and treated with TGFβ (10 ng/ml) for 24 h. n = 3 samples per group. Relative protein levels were shown after normalization to GAPDH. One-way ANOVA followed by Tukey’s multiple comparisons test. g , h Western blots ( g ) and quantitative results ( h ) of p-ALK5, p-SMAD2/3, and SMAD2/3 in HK-2 cells transfected with NEU1 and ALK5 160-200 plasmid for 24 h and treated with TGFβ (10 ng/ml) for 24 h. n = 3 samples per group. Relative protein levels were shown after normalization to GAPDH. One-way ANOVA followed by Tukey’s multiple comparisons test. i Co-immunoprecipitation of NEU1 and ALK5 in HK-2 cells stimulated with TGFβ (10 ng/ml) for 24 h. λpp, Lambda Protein Phosphatase. Two independent experiments were performed. j Western blots of ALK5 in HK-2 cells. siALK5-1, siALK5-2, and siALK5-3 were 3 different siRNA sequences. HK-2 cells were transduced with siALK5 for 48 h. Two independent experiments were performed. k – m mRNA levels of KIM1 ( k ) and protein level of p-ALK5, p-SMAD2/3, and SMAD2/3 ( l , m ) in HK-2 cells transduced with NEU1 full-length plasmid and ALK5 siRNA for 48 h. KIM1 mRNA normalized to GAPDH . Relative protein levels were shown after normalization to GAPDH. k n = 3 samples per group. l , m n = 3 samples per group. Two-way ANOVA followed by Tukey’s multiple comparisons test. n The proposed mechanisms of NEU1-mediated renal fibrosis. All statistic data were presented as mean ± SD. All tests were two-tailed.

Article Snippet: The primary antibodies used were as follows: anti-NEU1 (Proteintech, 67032-1-Ig, 1:1000 for IB, 1:100 for PLA), anti-NEU1(Santa Cruz, sc-166824, 1:100 for IP, 1:100 for IF, and 1:100 for IHC), anti-E-cadherin (CST, 3195s, 1:1000 for IB, 1:400 for IF, and 1:400 for ICH), anti-Vimentin (CST, 5741s, 1:1000 for IB, 1:400 for IF, 1:400 for ICH), anti-Snail (CST, 3879s, 1:1000 for IB and 1:400 for ICH), anti-Slug (CST, 9585s, 1:1000 for IB and 1:400 for ICH), anti-GAPDH (CST, 5174s, 1:1000 for IB), anti-α-smooth muscle actin (CST, 19245s, 1:1000 for IB and 1:400 for ICH), anti-HA-tag (Santa, sc-7392, 1:1000 for IB and 1:100 for IP), anti-Flag-tag (Affinity, T0053, 1:1000 for IB and 1:100 for IP), anti-ALK5 (Affinity, AF5347, 1:1000 for IB, 1:300 for IF, and 1:100 for IP, 1:100 for PLA), anti-p-ALK5 (Affinity, AF8080, 1:1000 for IB and 1:100 for IHC), anti-CD68 (CST, 97778, 1:200 for IHC), anti-p-NFκB (CST, 3037S, 1:200 for IHC), anti-Na/K ATPase (Abcam, ab254025, 1:200 for IF), anti-KIM1 (Cloud-Clone Corp, LAA785Mu81, 1:100 for IF), anti-Smad2 (CST, #5339, 1:1000 for IB), anti-Smad3 (CST, #9523, 1:1000 for IB), anti-p-Smad2 (CST, #3108, 1:1000 for IB), anti-p-Smad3 (CST, #9520, 1:1000 for IB), anti-LAMP1 (Abcam, ab24170, 1:200 for IHC), anti-PPCA (Abcam, ab184553, 1:1000 for IB), anti-biotinylated SNL lectin (Vector Labs, B-1305-2, 1:500 for IB).

Techniques: Transfection, Plasmid Preparation, Incubation, Western Blot, Quantitation Assay, Immunoprecipitation, Transduction, Two Tailed Test

Journal: Nature Communications

Article Title: Neuraminidase 1 promotes renal fibrosis development in male mice

doi: 10.1038/s41467-023-37450-8

Figure Lengend Snippet: a The interactions between 74 compounds with recombinant human NEU1 determined by surface plasmon resonance (SPR). K D , dissociation constant. The compound 3, 9, 11, 12, 18, 19, 20, 21, 41, 53, 58, 60, 61, 62, 67, 72, 73 have no K D value because they do not bind to NEU1. b , c The interaction between NEU1 and salvianolic acid B (SaB) ( b ) and salvianolic acid A (SaA) ( c ) was tested by SPR. The frequency response and fitting curves were displayed. Pearson’s test. d Scheme of the experimental approach. UUO, unilateral ureteral obstruction. e Hematoxylin and eosin (HE) and Masson’s trichrome staining from control (Ctrl) and SaA or SaB-treated mice 10 days after UUO. Scale bar, 50 mm. n = 3 mice per group. f Statistical results for interstitial collagen analyzed by Image Pro-Plus software. n = 3 mice per group. g – o the mRNA levels of kidney injury molecule 1 ( Kim1 , g ), EMT associate genes ( Snai1 and Snai2 , h , i ), inflammatory cytokines associate genes ( Tnfα , Il1 , and Il6 , j – l ) and extracellular matrix associate genes ( Vim , Col1a1 , and Col3a1 , m – o ) in kidney samples. All normalized to Gapdh . n = 6 samples per group. p Western blots ( p , left panel) and quantitative results ( p , right panel) of p-ALK5 (ser165), p-SMAD2/3, and SMAD2/3 in kidney from control (Ctrl) and SaA or SaB-treated mice 10 days after UUO. n = 3 mice per group. All statistic data were presented as mean ± SD, one-way ANOVA followed by Dunnett’s multiple comparisons test. All tests were two-sided.

Article Snippet: The primary antibodies used were as follows: anti-NEU1 (Proteintech, 67032-1-Ig, 1:1000 for IB, 1:100 for PLA), anti-NEU1(Santa Cruz, sc-166824, 1:100 for IP, 1:100 for IF, and 1:100 for IHC), anti-E-cadherin (CST, 3195s, 1:1000 for IB, 1:400 for IF, and 1:400 for ICH), anti-Vimentin (CST, 5741s, 1:1000 for IB, 1:400 for IF, 1:400 for ICH), anti-Snail (CST, 3879s, 1:1000 for IB and 1:400 for ICH), anti-Slug (CST, 9585s, 1:1000 for IB and 1:400 for ICH), anti-GAPDH (CST, 5174s, 1:1000 for IB), anti-α-smooth muscle actin (CST, 19245s, 1:1000 for IB and 1:400 for ICH), anti-HA-tag (Santa, sc-7392, 1:1000 for IB and 1:100 for IP), anti-Flag-tag (Affinity, T0053, 1:1000 for IB and 1:100 for IP), anti-ALK5 (Affinity, AF5347, 1:1000 for IB, 1:300 for IF, and 1:100 for IP, 1:100 for PLA), anti-p-ALK5 (Affinity, AF8080, 1:1000 for IB and 1:100 for IHC), anti-CD68 (CST, 97778, 1:200 for IHC), anti-p-NFκB (CST, 3037S, 1:200 for IHC), anti-Na/K ATPase (Abcam, ab254025, 1:200 for IF), anti-KIM1 (Cloud-Clone Corp, LAA785Mu81, 1:100 for IF), anti-Smad2 (CST, #5339, 1:1000 for IB), anti-Smad3 (CST, #9523, 1:1000 for IB), anti-p-Smad2 (CST, #3108, 1:1000 for IB), anti-p-Smad3 (CST, #9520, 1:1000 for IB), anti-LAMP1 (Abcam, ab24170, 1:200 for IHC), anti-PPCA (Abcam, ab184553, 1:1000 for IB), anti-biotinylated SNL lectin (Vector Labs, B-1305-2, 1:500 for IB).

Techniques: Recombinant, SPR Assay, Staining, Software, Western Blot

Journal: Nature Communications

Article Title: Neuraminidase 1 promotes renal fibrosis development in male mice

doi: 10.1038/s41467-023-37450-8

Figure Lengend Snippet: a Scheme of the experimental approach. The Neu1 CKO mice were treated with salvianolic acid B (SaB) at the indicated doses for 10 continuous days after being subjected to UUO surgery. b Representative gross appearance of kidneys (Scale bar, 2 mm), kidney cross-sections stained with HE (Scale bar, 50 μm), Masson’s trichrome (Scale bar, 50 μm), immunohistochemical staining with E-Cadherin and Snail (Scale bar, 50 μm). n = 3 mice per group. The red arrow indicates positive area. c Statistical results for interstitial collagen in b analyzed by Image Pro-Plus software. n = 3 mice per group. d Kim1 mRNA level. n = 3 mice per group. e , f Quantification of staining of E-Cadherin ( e ) and Snail1 ( f ). n = 3 mice per group. g , h mRNA levels of the indicated genes in kidneys determined by qRT-PCR. n = 3 mice per group. Dotted line represents the expression in sham control tissue. Gene expression levels were normalized to Gapdh . i Representative image of immunohistochemical staining with p-ALK5 (ser165) (Scale bar, 50 μm). n = 3 mice per group. j Quantification of staining of p-ALK5 (ser165). n = 3 mice per group. All data were presented as mean ± SD, one-way ANOVA followed by Tukey’s multiple comparisons test. All tests were two-tailed.

Article Snippet: The primary antibodies used were as follows: anti-NEU1 (Proteintech, 67032-1-Ig, 1:1000 for IB, 1:100 for PLA), anti-NEU1(Santa Cruz, sc-166824, 1:100 for IP, 1:100 for IF, and 1:100 for IHC), anti-E-cadherin (CST, 3195s, 1:1000 for IB, 1:400 for IF, and 1:400 for ICH), anti-Vimentin (CST, 5741s, 1:1000 for IB, 1:400 for IF, 1:400 for ICH), anti-Snail (CST, 3879s, 1:1000 for IB and 1:400 for ICH), anti-Slug (CST, 9585s, 1:1000 for IB and 1:400 for ICH), anti-GAPDH (CST, 5174s, 1:1000 for IB), anti-α-smooth muscle actin (CST, 19245s, 1:1000 for IB and 1:400 for ICH), anti-HA-tag (Santa, sc-7392, 1:1000 for IB and 1:100 for IP), anti-Flag-tag (Affinity, T0053, 1:1000 for IB and 1:100 for IP), anti-ALK5 (Affinity, AF5347, 1:1000 for IB, 1:300 for IF, and 1:100 for IP, 1:100 for PLA), anti-p-ALK5 (Affinity, AF8080, 1:1000 for IB and 1:100 for IHC), anti-CD68 (CST, 97778, 1:200 for IHC), anti-p-NFκB (CST, 3037S, 1:200 for IHC), anti-Na/K ATPase (Abcam, ab254025, 1:200 for IF), anti-KIM1 (Cloud-Clone Corp, LAA785Mu81, 1:100 for IF), anti-Smad2 (CST, #5339, 1:1000 for IB), anti-Smad3 (CST, #9523, 1:1000 for IB), anti-p-Smad2 (CST, #3108, 1:1000 for IB), anti-p-Smad3 (CST, #9520, 1:1000 for IB), anti-LAMP1 (Abcam, ab24170, 1:200 for IHC), anti-PPCA (Abcam, ab184553, 1:1000 for IB), anti-biotinylated SNL lectin (Vector Labs, B-1305-2, 1:500 for IB).

Techniques: Staining, Immunohistochemical staining, Software, Quantitative RT-PCR, Expressing, Two Tailed Test