Journal: Clinical Science (London, England : 1979)

Article Title: CCL5 deficiency aggravates acute DSS-induced colitis by restricting IL-33-induced formation of Tregs in intestinal tract

doi: 10.1042/CS20256734

Figure Lengend Snippet: ( A ) Immunoblotting of IL-33, ST2, forkhead box protein 3 (FOXP3), and JAK1/STAT5 signaling in intestinal CD4 + T cells of wildtype (WT) and CCL5 knockout ( Ccl5 -KO) mice 4 days after 7-day 2.5% dextran sulfate sodium salt (DSS) administration. ( B and C ) Mice received daily intraperitoneal injections of rIL-33 protein (10 ng/µL) at the onset of the DSS regimen. The intraperitoneal administration of different therapeutic agents continued for an additional 4 days after 7-day DSS treatment. Subsequently, the mice were killed, and the affected intestinal tissues were analyzed. Disease activity index (DAI) ( B ) and colon length ( C ) were monitored, n = 6 per group. ( D and E ) Representative H&E staining ( D ) of colon sections in mice from different treated groups (scale bars, 50 μm). Histological score ( E ) was quantified. ( F and G ) Representative FOXP3 staining of distal colon sections from rescued DSS-treated mice with indicated genotypes after 8-day rIL-33/PBS treatment ( F ; scale bars, 20 μm) and quantitative analysis ( G , n = 50). ( H and I ) Flow cytometric plots ( H ) of FOXP3 + CD4 + T cell population in intestines from rescued DSS-treated mice with indicated genotypes after 8-day rIL-33/PBS treatment and quantitative analysis ( I , n = 6). ( J ) Immunoblotting of FOXP3 and JAK1/STAT5 signaling in intestinal CD4 + T cells, respectively, from rescued DSS-treated mice with indicated genotypes after 8-day rIL-33/PBS treatment. The results are shown as the mean ± SEM (ns, nonsignificant; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001) of triplicate determination from three independent experiments, with statistical significance assessed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test.

Article Snippet: The following primary antibodies were utilized for the detection of target proteins: CCL5 (1:1000, Cell Signaling Technology Cat#: 36467, RRID:AB_3644243); FOXP3 (1:1000, Abcam, Cat#: ab215206, RRID: AB_2860568); IL33 (1:1000, Abcam, Mouse: Cat#: ab187060, RRID: AB_2894704/Human: Cat#: ab207737, RRID: AB_2827630); NF-κB p65 (1:1000, Abcam, Cat#: ab32536, RRID: AB_776751); Phospho-NF-κB p65 (Ser536) Monoclonal Antibody (1:1000, Thermo Fisher Scientific, Cat#: MA5-15160, RRID:AB_10983078); ST2 (1:1000, Invitrogen, Cat#: PA5-20077, RRID: AB_11156630); IkB alpha Monoclonal Antibody (1:1000, Thermo Fisher Scientific Cat#: MA5-15132, RRID:AB_10982641); Phospho-IkB alpha (Ser32, Ser36) Monoclonal Antibody (1:1000, Thermo Fisher Scientific Cat#: MA5-15224, RRID:AB_10981266); PI3K p85 alpha Monoclonal Antibody (1:1000, Thermo Fisher Scientific Cat#: MA1-74183, RRID:AB_2163452); Phospho-PI3K p85/p55 (Tyr458, Tyr199) Polyclonal Antibody (1:1000, Thermo Fisher Scientific Cat#: PA5-17387, RRID:AB_10985894); AKT Pan Polyclonal Antibody (1:1000, Thermo Fisher Scientific Cat#: 44-609G, RRID:AB_2533692); Phospho-AKT1 (Ser473) Monoclonal Antibody (1:1000, Thermo Fisher Scientific Cat#: 44-621G, RRID:AB_2533699); JAK1 Recombinant Rabbit Monoclonal Antibody (1:1000, Thermo Fisher Scientific Cat#: MA5-32780, RRID:AB_2810057); Phospho-JAK1 (Tyr1022, Tyr1023) Polyclonal Antibody (1:1000, Thermo Fisher Scientific Cat#: 44-422G, RRID:AB_2533648); STAT5A/B Polyclonal antibody (1:1000, Proteintech Cat#: 12071-1-AP, RRID:AB_2196933); Phospho-Stat5 (Tyr694) Antibody (1:1000, Cell Signaling Technology Cat#: 9351, RRID:AB_2315225); β-actin (1:10,000; Abcam, Cat#: ab6276, RRID: AB_2223210).

Techniques: Western Blot, Knock-Out, Activity Assay, Staining

Journal: bioRxiv

Article Title: Atlas of Lysosomal Aging Reveals a Molecular Clock of Storage Disorder-Associated Metabolites

doi: 10.1101/2025.09.25.678303

Figure Lengend Snippet: (A-L) Immunoblots of tissue lysates and lysosomal fractions from the indicated tissues of male mice (A-K), and female mice (L), probed for markers of lysosomes and other cellular compartments. A representative sample from each group is shown. Lamp1 – Lysosomal-associated membrane protein 1, marker of the lysosomal membrane; Lamp2 – Lysosomal-associated membrane protein 2, marker of the lysosomal membrane; CtsD – cathepsin D, marker of the lysosomal lumen; CtsL – cathepsin L, marker of the lysosomal lumen (probed in WAT), CtsB – cathepsin B, marker of the lysosomal lumen (probed in the muscle); CS – citrate synthase, mitochondrial marker; SDHA – succinate dehydrogenase complex flavoprotein subunit A, mitochondrial marker (probed in uterus); CALR – calreticulin, marker of the endoplasmic reticulum; GAPDH – Glyceraldehyde 3-phosphate dehydrogenase, cytoplasmic marker; USO1 – General vesicular transport factor p115, Golgi apparatus marker; ACOX1 – acyl-CoA oxidase 1, peroxisomal marker. Next to each immunoblot, the corresponding protein quantification is shown for young (n = 2) and old (n = 2) mock purification samples (LysoTag -), as well as young (n = 5) and old (n = 5) lysosomal fractions (LysoTag +). For the uterus samples, n = 2 mice per group. Note that in (A), the Lamp1, CtsD, and CS blots are identical to those in the abbreviated panel contained in .

Article Snippet: The following primary antibodies were used: Lamp1 (DSHB, 1D4B; RRID: AB_2134500), Lamp2 [ABL-93] (Abcam, ab25339; RRID: AB_470455), CtsD (R&D, AF1029; RRID: AB_2087094), CtsL (R&D, AF1515; RRID: AB_2087690), CtsB (CST, 31718S; RRID: AB_2687580), Citrate Synthase (CST, 14309S; RRID: AB_2665545), CALR (CST, 12238S; RRID: AB_2688013; and Abcam, ab92516; RRID: AB_10562796 for detection in rat samples), GAPDH (CST, 2118L; RRID: AB_561053), USO1 (Proteintech, 13509-1-AP; RRID: AB_2257094), ACOX1 (Abcam, ab184032; RRID: AB_2904240), NPC1 (Abcam, ab134113; RRID: AB_2734695), SDHA (CST, 11998S; RRID: AB_2750900), Vinculin (CST, 13901S; RRID: AB_2728768).

Techniques: Western Blot, Membrane, Marker, Purification

Journal: bioRxiv

Article Title: Atlas of Lysosomal Aging Reveals a Molecular Clock of Storage Disorder-Associated Metabolites

doi: 10.1101/2025.09.25.678303

Figure Lengend Snippet: (A) Schematic of the experimental setup for the isolation of lysosomes from tissues of wildtype animals in vivo using an antibody against the lysosome-specific anchor protein p18. (B) Lysosomal fractions and whole tissue lysates from the brains of young (2-3 months old; n = 8) and old (24 months old; n = 8) wildtype mice were analyzed by immunoblotting for markers of lysosomes and other cellular compartments (left), and by targeted liquid chromatography and mass spectrometry for the quantification of cystine and glycerophosphodiesters (right). Lamp1 – Lysosomal-associated membrane protein 1, marker of the lysosomal membrane; Lamp2 – Lysosomal-associated membrane protein 2, marker of the lysosomal membrane; CtsD – cathepsin D, marker of the lysosomal lumen; CS – citrate synthase, mitochondrial marker; CALR – calreticulin, marker of the endoplasmic reticulum; GAPDH – Glyceraldehyde 3-phosphate dehydrogenase, cytoplasmic marker; USO1 – General vesicular transport factor p115, Golgi apparatus marker; ACOX1 – acyl-CoA oxidase 1, peroxisomal marker. (C) Lysosomal fractions and whole tissue lysates from the hearts of young (2-3 months old; n = 8) and old (24 months old; n = 9) wildtype mice were analyzed by immunoblotting for markers of lysosomes and other cellular compartments (left), and by targeted liquid chromatography and mass spectrometry for the quantification of cystine and glycerophosphodiesters (right). Markers probed as in (B). (D) Lysosomal fractions and whole tissue lysates from the brains of young (4 months old; n = 8) and old (25 months old; n = 8) wildtype rats were analyzed by immunoblotting for markers of lysosomes and other cellular compartments (left), and by targeted liquid chromatography and mass spectrometry for the quantification of cystine and glycerophosphodiesters (right). NPC1 – Lysosomal-associated membrane protein 1, marker of the lysosomal membrane; CtsL – cathepsin L, marker of the lysosomal lumen; SDHA – succinate dehydrogenase complex flavoprotein subunit A, mitochondrial marker; CALR – calreticulin, marker of the endoplasmic reticulum; Vinculin, cytoplasmic marker; USO1 – General vesicular transport factor p115, Golgi apparatus marker, ACOX1 – acyl-CoA oxidase 1, peroxisomal marker. (B-D) Data are presented as mean ± SEM. p-values were determined using two-tailed t-tests.

Article Snippet: The following primary antibodies were used: Lamp1 (DSHB, 1D4B; RRID: AB_2134500), Lamp2 [ABL-93] (Abcam, ab25339; RRID: AB_470455), CtsD (R&D, AF1029; RRID: AB_2087094), CtsL (R&D, AF1515; RRID: AB_2087690), CtsB (CST, 31718S; RRID: AB_2687580), Citrate Synthase (CST, 14309S; RRID: AB_2665545), CALR (CST, 12238S; RRID: AB_2688013; and Abcam, ab92516; RRID: AB_10562796 for detection in rat samples), GAPDH (CST, 2118L; RRID: AB_561053), USO1 (Proteintech, 13509-1-AP; RRID: AB_2257094), ACOX1 (Abcam, ab184032; RRID: AB_2904240), NPC1 (Abcam, ab134113; RRID: AB_2734695), SDHA (CST, 11998S; RRID: AB_2750900), Vinculin (CST, 13901S; RRID: AB_2728768).

Techniques: Isolation, In Vivo, Western Blot, Liquid Chromatography, Mass Spectrometry, Membrane, Marker, Two Tailed Test

Journal: bioRxiv

Article Title: Atlas of Lysosomal Aging Reveals a Molecular Clock of Storage Disorder-Associated Metabolites

doi: 10.1101/2025.09.25.678303

Figure Lengend Snippet: (A) Immunoblot analyses of mouse liver tissue lysates and lysosomal fractions using a panel of antibodies against lysosomal membrane proteins, along with rabbit IgG for a mock purification. Samples were probed for markers of lysosomes and other cellular compartments as in . (B) Heatmap representation of log 2 -fold changes in a panel of targeted metabolites detected in lysosomal fractions obtained via anti-p18 immunoisolation from mouse liver (n = 3), heart (n = 4) and brain (n = 3), relative to mock purifications using isotype-specific IgG (n = 2 per tissue). Crossed tiles indicate metabolites that were not detected or not enriched above the mock purification. (C) Immunoblots of rat liver tissue lysates and lysosomal fractions obtained using a panel of antibodies against lysosomal membrane proteins, along with isotype-specific rabbit IgG for a mock purification. Samples were probed for markers of lysosomes and other cellular compartments: NPC1 – Lysosomal-associated membrane protein 1, marker of the lysosomal membrane; CtsL – cathepsin L, marker of the lysosomal lumen; SDHA – succinate dehydrogenase complex flavoprotein subunit A, mitochondrial marker, mitochondrial marker; CALR – calreticulin, marker of the endoplasmic reticulum; Vinculin, cytoplasmic marker; USO1 – General vesicular transport factor p115, Golgi apparatus marker. (D) Heatmap representation of log 2 -fold changes in a panel of targeted metabolites detected in lysosomal fractions obtained via anti-p18 immunoisolation from rat brain, relative to mock purifications with isotype specific IgG (n = 2).

Article Snippet: The following primary antibodies were used: Lamp1 (DSHB, 1D4B; RRID: AB_2134500), Lamp2 [ABL-93] (Abcam, ab25339; RRID: AB_470455), CtsD (R&D, AF1029; RRID: AB_2087094), CtsL (R&D, AF1515; RRID: AB_2087690), CtsB (CST, 31718S; RRID: AB_2687580), Citrate Synthase (CST, 14309S; RRID: AB_2665545), CALR (CST, 12238S; RRID: AB_2688013; and Abcam, ab92516; RRID: AB_10562796 for detection in rat samples), GAPDH (CST, 2118L; RRID: AB_561053), USO1 (Proteintech, 13509-1-AP; RRID: AB_2257094), ACOX1 (Abcam, ab184032; RRID: AB_2904240), NPC1 (Abcam, ab134113; RRID: AB_2734695), SDHA (CST, 11998S; RRID: AB_2750900), Vinculin (CST, 13901S; RRID: AB_2728768).

Techniques: Western Blot, Membrane, Purification, Marker