cleavage buffer  (Verlag GmbH)

Journal: The Journal of Biological Chemistry

Article Title: Divergent pathways of surfactant protein C maturation for disease-associated isoforms

doi: 10.1016/j.jbc.2026.111252

Figure Lengend Snippet: Furin-like pro-protein convertases are candidate enzymes for initial SP-C cleavage. A and B , Western blot analysis and quantitation of c-term SP-C cleavage ( arrowheads ) in MLE-12 cells cotreated with DC1 (dicoumarol-related pan-proprotein convertase inhibitor) and doxycycline to induce the expression of GFP-SPC WT for 9 h. C and D , immunoblot and quantitation of C-terminal SP-C cleavage after overnight DC1 treatment in primary murine AT2 isolated from SP-C WT mice shows similar inhibition of SP-C processing. E , immunofluorescence of GFP-SPC WT MLE-12 cells cotreated with doxycycline and DC1 for 9 h, fixed and stained with GM130 (Golgi marker). F , immunofluorescence of GFP-SPC WT after 6 h of doxycycline-induction ( green ) shows colocalization with furin ( magenta ) in the perinuclear region of MLE12 cells. Hoechst stains nuclei. Scale bars represent 10 μm. G , GFP-pulldown in MLE12 cells using GFP-nanotrap or Control Binding-nanotrap beads shows interaction of mature furin with GFP-tagged SP-C. H , furin cleavage assay performed with SP-C pro-translation product as the in vitro substrate in the presence and absence of decanoyl-RVKR-CMK. I , immunoblot after GFP-pulldown enrichment following 9 h of doxycycline showing SP-C processing in MLE-12 cells transduced with individual pre-profragment of PC7 or furin or IRES empty vector control. J , immunofluorescence of GFP-SPC WT after overnight of doxycycline-induction and decanoyl-RVKR-CMK furin inhibitor stained for LAMP3. K , immunoblot of GFP-SPC WT expressing MLE-12 cells after overnight treatment of doxycycline and decanoyl-RVKR-CMK. L , quantification ratio of SP-C cleaved intermediate relative to pro-form SP-C in each condition. SP-C, surfactant protein C; MLE-12, mouse lung epithelial-12; AT2, alveolar type II.

Article Snippet: Afterward, beads were recovered by centrifugation, washed three times in washing buffer, and then incubated with 75 μl of furin cleavage buffer (20 mM Hepes, 1 mM CaCl 2 , 0.2 mM β-mercaptoethanol, 0.1% Triton X-100; pH 7.5) supplemented with 1 U furin (#P8077S, NEB) for 1 h at 37 °C.

Techniques: Western Blot, Quantitation Assay, Expressing, Isolation, Inhibition, Immunofluorescence, Staining, Marker, Control, Binding Assay, Cleavage Assay, In Vitro, Transduction, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Divergent pathways of surfactant protein C maturation for disease-associated isoforms

doi: 10.1016/j.jbc.2026.111252

Figure Lengend Snippet: WT surfactant protein C (SP-C) undergoes ordered trafficking through the Golgi and trans-Golgi network (TGN), where a furin-like proprotein convertase mediates initial C-terminal cleavage prior to routing through multivesicular bodies to lamellar bodies for maturation and secretion. In contrast, the disease-associated SP-C I73T mutant diverges early from this pathway (denoted by asterisk ; ∗), and accumulates at the plasma membrane. SP-C, surfactant protein C.

Article Snippet: Afterward, beads were recovered by centrifugation, washed three times in washing buffer, and then incubated with 75 μl of furin cleavage buffer (20 mM Hepes, 1 mM CaCl 2 , 0.2 mM β-mercaptoethanol, 0.1% Triton X-100; pH 7.5) supplemented with 1 U furin (#P8077S, NEB) for 1 h at 37 °C.

Techniques: Mutagenesis, Clinical Proteomics, Membrane