Journal: Biomolecules

Article Title: Upregulation of Sortilin, a Lysosomal Sorting Receptor, Corresponds with Reduced Bioavailability of Latent TGFβ in Mucolipidosis II Cells

doi: 10.3390/biom10050670

Figure Lengend Snippet: Increased intracellular latent TGFβ1 in ML-II fibroblasts is localized within dense, detergent-insoluble lysosomal fractions. ( A ) Western blot analysis of pooled Percoll gradient fractions from control and ML-II cellular lysates (n = 4). SDS-PAGE-resolved fractions were subjected to analysis using antibodies against markers for lysosomes (LAMP-2), Golgi (GS28) and ER (ERp29) as well as LAP. Pool I, dense membranes (lysosomes); pool II, intermediate density organelles; pool III, ER and Golgi membranes. ( B ) Western blots of human control and ML-II cells fractionated into deoxycholate (DOC)-soluble (S) and –insoluble (I) fractions (n = 4). Along with concentrated media (M) fractions, these pools were resolved by non-reducing (NR) SDS-PAGE and immunoblotted with antibodies against human LAP or LTBP-1. The upper and lower arrowheads indicate the position of the LLC and free LTBP-1, respectively ( C ) Luciferase assays (n = 3) of heat-treated deoxycholate-insoluble fractions that were applied to mink lung TGFβ1 reporter cells; luciferase activity was measured in relative luminescence units, ** p ≤ 0.01.

Article Snippet: Goat antisera against human LAP and monoclonal antibody against human LTBP was purchased from R & D systems (USA), while the monoclonal antibody γ-tubulin was obtained from Sigma.

Techniques: Western Blot, SDS Page, Luciferase, Activity Assay

Journal: Biomolecules

Article Title: Upregulation of Sortilin, a Lysosomal Sorting Receptor, Corresponds with Reduced Bioavailability of Latent TGFβ in Mucolipidosis II Cells

doi: 10.3390/biom10050670

Figure Lengend Snippet: Impaired secretion and increased insolubility and of latent TGFβ1 and reduced TGFβ1 signaling, are also noted in feline ML-II fibroblast-like synoviocytes. ( A ) Western blots of feline control and ML-II cells fractionated into deoxycholate-soluble (S) and –insoluble (I) fractions (n = 3). These fractions and concentrated media (M) from cultures, were resolved by non-reducing (NR) SDS-PAGE and immunoblotted with an antibody against human LAP. ( B ) Western blot analysis of control and ML-II feline fibroblast lysates using antibodies against Smad 2/3 and phosphorylated Smad 2. ( C ) Quantification of the relative intensity of phosphorylated Smad 2 vs. Smad 2/3 protein in control and ML-II cells (n = 3), ** p ≤ 0.01.

Article Snippet: Goat antisera against human LAP and monoclonal antibody against human LTBP was purchased from R & D systems (USA), while the monoclonal antibody γ-tubulin was obtained from Sigma.

Techniques: Western Blot, SDS Page

Journal: Biomolecules

Article Title: Upregulation of Sortilin, a Lysosomal Sorting Receptor, Corresponds with Reduced Bioavailability of Latent TGFβ in Mucolipidosis II Cells

doi: 10.3390/biom10050670

Figure Lengend Snippet: Sortilin-1 protein and transcript levels are elevated in ML-II but not ML-III, fibroblasts. Western blots for sortilin-1 in control and ML-II human fibroblasts ( A ) and feline fibroblast-like synoviocytes ( B ) using a mouse monoclonal antibody against human sortilin-1 (n = 3). ( C ) Sortilin-1 Western blot in WT and GNPTAB-/- (KO) HeLa cells (n = 2). ( D ) Sortilin-1 Western blot of DOC-soluble and –insoluble fractions of human control and ML-II fibroblasts (n = 3). ( E ) Western blot of control and ML-II feline chondrocytes (FC) and fibroblast-like synoviocytes (FLS) as well as control and ML-II human fibroblasts (HF) using a polyclonal antibody against human cathepsin D (n = 2). ( F ) Immunoblots for sortilin-1 and LAP in control, ML-III and ML-II human fibroblasts. Note the correlation of increased sortilin levels and accumulation of LAP in ML-II but not ML-III cells. ( G ) Representative reverse transcription-polymerase chain reaction (RT-PCR) analysis of sortilin-1 transcripts. Ribosomal Protein L4 (RPL4) transcript abundance is shown as a loading control. ( H ) Quantification of the intensity of SORT1 relative to RPL4 (n = 3), *** p ≤ 0.001.

Article Snippet: Goat antisera against human LAP and monoclonal antibody against human LTBP was purchased from R & D systems (USA), while the monoclonal antibody γ-tubulin was obtained from Sigma.

Techniques: Western Blot, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction