Journal: bioRxiv

Article Title: Heparan sulfate promotes autoactivation of pro-Cathepsin K by destabilizing the propeptide-catalytic domain interaction

doi: 10.64898/2026.01.18.700217

Figure Lengend Snippet: Mouse femur section was co-stained with anti-CtsK (green), HS20 (purple) and anti-LAMP2 (red) and visualized with anti-rabbit Alexa488, anti-human Alexa594, and anti-rat Alexa647 secondary antibodies, respectively. Area shown is the cortical bone. The approximate boundary of the bone is shown with white dashed line. A blood vessel within the bone is indicated with yellow dashed line. Red blood cells in the vessel shown strong green autofluorescence. A closeup view of the large osteoclast is shown in the right bottom panel. Another osteoclast (likely only the tip of the osteoclast) is indicated with a yellow arrow in the merged image. Nuclei of several osteocytes are indicated with white arrow head. Images representative of three separate experiments.

Article Snippet: Following deparaffinization and citric acid-based antigen retrieval (pH7), sectioned were blocked and incubated overnight at 4 °C with the following primary antibodies: 0.2 μg/ml rabbit anti-mCtsK polyclonal antibody (described previously) , 1ug/ml human anti-HS mAb (HS20, from Bio X cell) , and 1ug/ml Rat anti-mouse Lamp2 (GL2A7, Developmental Studies Hybridoma Bank).

Techniques: Staining

Journal: Acta Neuropathologica Communications

Article Title: Pathobiology of the autophagy-lysosomal pathway in the Huntington’s disease brain

doi: 10.1186/s40478-025-02131-8

Figure Lengend Snippet: Correlation between marker expression levels and HD progression stages across different brain regions, grouped by marker category. The heatmap illustrates Spearman’s rank correlation coefficients (ρ) between the relative mRNA expression levels from qPCR of selected molecular markers and Huntington’s disease (HD) severity. Analyses were performed on human post-mortem brain tissue from control (Ctl) and HD patients (grades HD2, HD3, HD4), with severity numerically encoded for correlation (Ctl = 0, HD2 = 1, HD3 = 2, HD4 = 3). Correlations are shown for STR, CTX, and CBM). Markers are grouped into functional categories, displayed from top to bottom: Induction Markers (Autophagy induction): ATG7, LC3, p62. Lysosomal Markers (Lysosomal biogenesis, hydrolases and structural elements): TFEB, TFE3, CTSB, CTSD, HEXA, LAMP1, LAMP2. Cellular Markers (cellular processes): ENO2, TUBB3, GFAP, AIF1, MBP. The color of each cell represents the Spearman ρ value, with a continuous gradient from blue (strong negative correlation, ρ = −1) through white (no correlation, ρ = 0) to red (strong positive correlation, ρ = + 1). Nominal p-values for the correlations are indicated by asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies for immunohistochemistry (IHC), western blotting (WB): The following primary antibodies were used in this study. (1) from Cell Signaling Technology: tHTT rabbit mAb (clone D7F7, #5656, targeting residues surrounding Pro1220 of human HTT and detecting total HTTs), p70S6K pAb (#9202), p-p70S6K (T389) pAb (#9205), ULK1 pAb (#4773), p-ULK1 (S757) pAb (#6888, #14202; detecting S757 or S758 of mouse or human ULK1, respectively), ATG5 rabbit mAb (#12994), ATG7 pAb (#2631), ATG13 rabbit mAb (#13273), p-ATG13 (S355) rabbit mAb (#26839), VPS34 rabbit mAb (#81453), TRAF6 rabbit mAb (#8028), Calnexin rabbit mAb (#2679). (2) from Millipore-Sigma: ntHTT mAb (N-Terminus-specific, mEM48, #MAB5374, preferentially recognizing aggregated HTT) , ATG5 pAb (#ABC14), K48- or K63-specific ubiquitin mAb (#05–1307, #05–1308, respectively), βIII-tubulin mAb (#SAB4700544), β–actin mAb (#A1978). (3) from other vendors: BECN1 mAb (BD Biosciences, #612113); LC3 pAb (Novus Biologics, #NB100-2220), ATG9 (Novus Biologics, #B-110–56893); p62 mAb (BD Biosciences, #610832) or C-term-specific p62 Guinea Pig pAb (Progen Biotechnik, #C-1620); total ubiquitin pAb (Dako Agilent, #Z0458), LAMP1 or LAMP2 rat mAb (Developmental Studies Hybridoma Bank, University of Iowa, #H4A3 or #H4B4, respectively); CTSD sheep pAb (D-2–3, in-house made) ( ); CTSD pAb (Scripps Laboratories, #RC245), CTSD mAb (CD1.1, in-house made) ( ); CTSB pAb (Cortex Biochemicals, #CR6009RP), CTSB goat pAb (Neuromics, #GT15047).

Techniques: Marker, Expressing, Control, Functional Assay

Journal: bioRxiv

Article Title: Cisplatin and Copper Induce Distinct Endocytic Pathways of Copper Transporter-1 and Elicits Unique Response on Copper Homeostasis Proteins

doi: 10.1101/2025.11.03.686439

Figure Lengend Snippet: Fluorescence imaging showing fraction of CTR1 (green) localized with respective endocytic vesicles (red) while DAPI (blue) marks the nucleus at copper (50 µM) and CDDP (25µM & 50µM). The Scale bar represents 10 microns. (A) Immunofluorescence image showing fraction of Ctr1 (green) with early endosomal marker EEA1 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-EEA1. (B) Immunofluorescence image showing fraction of Ctr1 (green) with retromer controlled Recycling endosomal vesicle VPS35 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-VPS35. (C) Immunofluorescence image showing fraction of Ctr1 (green) with late endosomal marker Rab7 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab7. (D) Immunofluorescence image showing fraction of Ctr1 (green) with retromer late endosomal marker Rab9 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab9. (E) Immunofluorescence image showing fraction of Ctr1 (green) with lysosomal resident protein Lamp2 (Red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Lamp2. (F) Quantitative analysis showing fraction of CTR1 residing in different endocytic vesicles upon 1 hr. treatment of copper (100 µM) and CDDP (100 µM) respectively graphically represented using a Box and Whisker plot with jitter points (n>50). The box represents the 25 to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5 × interquartile range (IQR) from the first and third quartile. ∗ p < 0.05, ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: The primary antibodies utilized in immunofluorescence studies are as follows: Rabbit anti-FLAG M2 ( :600,CST #14793), Rabbit Ctr1(1:800,# ab129067 Abcam), Mouse Na/K ATPase ( :600,#MA3-929 Invitrogen), Mouse VPS35 ( :500,#sc-374372, Santa Cruz Biotechnology), Mouse Rab7 ( :50,#sc-376362, Santa Cruz Biotechnology), Mouse Lamp2 ( :150, #H4B4 DSHB), Mouse Rab9 ( :100, #MA3-06 Invitrogen), Mouse EEA1(1:100 #610457, 1:400; BD Biosciences).The Secondary antibodies utilized in immunofluorescence studies are as follows: Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 ( :2000, #A-21206 Invitrogen), Donkey anti-Mouse Alexa 555 (#A-32773, 1:2000; Invitrogen).

Techniques: Fluorescence, Imaging, Immunofluorescence, Marker, Whisker Assay

Journal: bioRxiv

Article Title: Cisplatin and Copper Induce Distinct Endocytic Pathways of Copper Transporter-1 and Elicits Unique Response on Copper Homeostasis Proteins

doi: 10.1101/2025.11.03.686439

Figure Lengend Snippet: (A) Immunofluorescence image showing fraction of Ctr1 (green) with early endosomal marker EEA1 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-EEA1. (B) Immunofluorescence image showing fraction of Ctr1 (green) with retromer controlled Recycling endosomal vesicle VPS35 (red) in HEK293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-VPS35. (C) Immunofluorescence image showing fraction of Ctr1 (green) with late endosomal marker Rab7 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab7. (D) Immunofluorescence image showing fraction of Ctr1 (green) with retromer late endosomal marker Rab9 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab9. (E) Immunofluorescence image showing fraction of Ctr1 (green) with lysosomal resident protein Lamp2 (Red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Lamp2. (F) Quantitative analysis showing fraction of CTR1 residing in different endocytic vesicles upon 1 hr. treatment of copper (100 µM) and CDDP (100 µM) respectively graphically represented using a Box and Whisker plot with jitter points (n>50). The box represents the 25 to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5 × interquartile range (IQR) from the first and third quartile. ∗ p < 0.05, ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: The primary antibodies utilized in immunofluorescence studies are as follows: Rabbit anti-FLAG M2 ( :600,CST #14793), Rabbit Ctr1(1:800,# ab129067 Abcam), Mouse Na/K ATPase ( :600,#MA3-929 Invitrogen), Mouse VPS35 ( :500,#sc-374372, Santa Cruz Biotechnology), Mouse Rab7 ( :50,#sc-376362, Santa Cruz Biotechnology), Mouse Lamp2 ( :150, #H4B4 DSHB), Mouse Rab9 ( :100, #MA3-06 Invitrogen), Mouse EEA1(1:100 #610457, 1:400; BD Biosciences).The Secondary antibodies utilized in immunofluorescence studies are as follows: Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 ( :2000, #A-21206 Invitrogen), Donkey anti-Mouse Alexa 555 (#A-32773, 1:2000; Invitrogen).

Techniques: Immunofluorescence, Marker, Whisker Assay

Journal: bioRxiv

Article Title: Cisplatin and Copper Induce Distinct Endocytic Pathways of Copper Transporter-1 and Elicits Unique Response on Copper Homeostasis Proteins

doi: 10.1101/2025.11.03.686439

Figure Lengend Snippet: (A) Magic red, a substrate of Cathepsin B which signifies lysosomal activity showed via immunofluorescence images in Hek293T cell line. Red dots indicate cathepsin positive lysosomes in Copper and CDDP treated condition. (B) Cathepsin B activity measured using Corrected total cell fluorescence (CTCF) analysis and plotted (C) Size of cathepsin positive lysosomes analyzed and plotted. (D) Lysosensor Green, dye measuring change in lysosomal P.H used to measure acidic characteristics of Lysosome via Flow cytometry(n=3). (E) Expression of Lamp2, a lysosomal membrane resident glycoprotein associate with lysosomal number and function showed by Western blot. (F) Abundance of Lamp2 in Hek293T cells analyzed using Non-Parametric T test and plotted. (n=3) (G) Number of acidic lysosomal compartments measured using Lysotracker via Flow cytometry analyzed and plotted . (n=3)

Article Snippet: The primary antibodies utilized in immunofluorescence studies are as follows: Rabbit anti-FLAG M2 ( :600,CST #14793), Rabbit Ctr1(1:800,# ab129067 Abcam), Mouse Na/K ATPase ( :600,#MA3-929 Invitrogen), Mouse VPS35 ( :500,#sc-374372, Santa Cruz Biotechnology), Mouse Rab7 ( :50,#sc-376362, Santa Cruz Biotechnology), Mouse Lamp2 ( :150, #H4B4 DSHB), Mouse Rab9 ( :100, #MA3-06 Invitrogen), Mouse EEA1(1:100 #610457, 1:400; BD Biosciences).The Secondary antibodies utilized in immunofluorescence studies are as follows: Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 ( :2000, #A-21206 Invitrogen), Donkey anti-Mouse Alexa 555 (#A-32773, 1:2000; Invitrogen).

Techniques: Activity Assay, Immunofluorescence, Fluorescence, Flow Cytometry, Expressing, Membrane, Western Blot