pngase f  (New England Biolabs)

Journal: The Journal of Biological Chemistry

Article Title: Enzymatic basis of branching and extension of O -Man glycans for keratan sulfate biosynthesis

doi: 10.1016/j.jbc.2026.111140

Figure Lengend Snippet: The levels of KS in GnT-IX-KO and POMGNT1-KO mouse brains . A , schematic diagram of phosphacan. O -Man glycans, N -glycans, O -GalNAc glycans (mucin-type glycans), chondroitin sulfate (CS), and keratan sulfate (KS) are present on phosphacan. B , Phosphacan-Fc (Phos-Fc) and GnT-IX were expressed in HEK293 C1GALT1 KO cells. ( Left ) Cell lysates were western blotted for GnT-IX and GAPDH. ( Right ) Phosphacan was purified from the media using Dynabeads protein G. After treatment of phosphacan with ChABC, PNGaseF, and sialidase, proteins were separated by SDS-PAGE and blotted with anti-human IgG. C , schematic model of the reactions catalyzed by POMGNT1 and GnT-IX. D , Phosphacan was expressed in HMCB WT and POMGNT1-KO cells (clone #3 in ) and purified from the media. After treatment of phosphacan with ChABC, proteins were separated by SDS-PAGE and blotted with anti-human IgG and R-10G. E , representative glycan structures recognized by 5D4 and R-10G antibodies and LEL lectin. F , blotting of proteins in the soluble fractions from GnT-IX ( Mgat5b ) heterozygous and KO mouse brains (postnatal day 0 [P0]) with anti-phosphacan, 5D4, R-10G, anti-GAPDH or LEL lectin. G , signal intensities of the band corresponding to the position of phosphacan in 5D4, R-10G, and LEL blots in ( F ) were quantified, and adjusted for GAPDH. The relative levels are shown as a graph (n = 3, mean ± SD, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, unpaired t test). H , blotting of proteins in the soluble fractions from WT and POMGNT1-KO mouse brains (female, 4-week-old) with anti-phosphacan, R-10G, or anti-GAPDH.

Article Snippet: Samples were then incubated with water or PNGaseF (NEB, P0704) at 37 °C for 2 h. The samples were then mixed with 5 × Laemmli sample buffer and incubated at 95 °C for 5 min. For sialidase treatment, Clostridium perfringens α2-3,6,8 sialidase (New England Biolabs, P0720) was added simultaneously with PNGaseF.

Techniques: Western Blot, Purification, SDS Page, Glycoproteomics

Journal: bioRxiv

Article Title: OSTM1 is a ubiquitin E3 ligase that suppresses B-cell malignancy by activating the cAMP/PKA/CREB pathway

doi: 10.64898/2026.01.23.701155

Figure Lengend Snippet: (A) sg Ostm1 and sgControl Ba/F3 cells were subjected to TMT-MS. Volcano plot (log₂ fold change vs. –log₁₀ p-value) was generated using VolcaNoseR. Dashed lines indicate significance cutoffs. Selected proteins involved in hematopoeitic function and differentiation are highlighted in red. (B) KEGG pathway enrichment analysis of proteins involved in sg Ostm1 Ba/F3 cells with-log10(FDR) used as the significance metric. (C) Venn diagram depicting the overlap between proteins enriched in sg Ostm1 cells identified by TMT-MS and OSTM1-interacting partners identified by BioID in Ba/F3 cells. The table shows BioID enrichment scores for Pde3b. (D and E) His-tagged PDE3B and Flag-tagged OSTM1 were co-transfected into HEK293T cells, in the presence or absence of MG132 (10 µM). Co-immunoprecipitation (Co-IP) was performed using anti-His (D) or anti-Flag (E) antibodies, followed by IB. (F) Ostm1 was silenced in Ba/F3 cells, and three independent cell clones were probed for Pde3b by IB. (G) qRT-PCR analysis of sg Ostm1 cell clones showing that Pde3b mRNA levels were not affected by Ostm1 silencing. (H) OSTM1-Flag was stably reconstituted in sg Ostm1 Ba/F3 cell clone #3, and Pde3b was probed by IB. (I) sgControl and sg Ostm1 Ba/F3 cells were treated with cycloheximide (CHX, 100 µg/ml) for the indicated hours. Pde3b was probled by IB and quantified using ImageJ. Pde3b half-life was calculated based on densitometry. Total ubiquitylated proteins were probed as an indicator of global protein turnover following CHX tratment. (J) sgControl and sg Ostm1 Ba/F3 cells were treated with MG132 (10 µM) or chloroquine (100 µM) for indicated hours, in the presence of CHX (100 µg/ml). Pde3b stability was determined by IB. Ubiquitin and Lc3b were probed to confirm the effectiveness of MG132 and CQ treatment. The Pde3b/Gapdh ratio is shown below the blots. (K) sgControl and sg Ostm1 Ba/F3 cells were treated with indicated concenrations of MG132 for 8 h, and Pde3b was detected by IB. (L) PDE3B-His was expressed in sgControl or sg Ostm1 Ba/F3 cells. IP with PDE3B antibody was performed, followed by IB with indicated antibodies. PDE3B-His ubiquitination was detected in sgControl cells and reduced in sg Ostm1 cells. (M) OSTM1-Flag and PDE3B-His were transfected into HEK293T cells, individually or together, along with His-ubiquitin. Cells were treated with MG132 (10 µM) for 8 h prior to IP with PDE3B antibody and IB. (N) OSTM1-Flag and PDE3B-His were expressed separately in HEK293T cells. Cell lysates were used individually or combined for in vitro ubiquitylation assays. OSTM1-Flag promoted both its auto-ubiquitylation and PDE3B ubiquitylation. (O) Spearman correlation analysis of OSTM1 and PDE3B protein abundance across BCL cell lines from different subtypes using CCLE proteomic data. (P) OSTM1-Flag was stably expressed in indicated BCL cell lines. Flag pull-down assays were performed, and interacting proteins were analyzed by IB. (Q and R) OSTM1 was silenced in SU-DHL-5 ( Q ) and ARH-77 ( R ) cell lines. Two clones per cell line were probed for PDE3B by IB. (S) sgControl and sg OSTM1 ARH-77 cells were treated with CHX (100 µg/ml). PDE3B was probed by IB, and its half-life was calculated based on ImageJ densitometric quantification.

Article Snippet: OSTM1-Flag expressing cell lysates underwent in-vitro PNGase-F treatment (NEB, P0704) under non-denaturing conditions following manufacturer’s protocol.

Techniques: Generated, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Clone Assay, Quantitative RT-PCR, Stable Transfection, Ubiquitin Proteomics, In Vitro, Quantitative Proteomics

Journal: bioRxiv

Article Title: OSTM1 is a ubiquitin E3 ligase that suppresses B-cell malignancy by activating the cAMP/PKA/CREB pathway

doi: 10.64898/2026.01.23.701155

Figure Lengend Snippet: (A) OCI-LY-18 cells stably expressing OSTM1-Flag were treated with tunicamycin (Tuni, 5 µg/ml), brefeldin A (BFA, 3 µg/ml), and thapsigargin (Thap, 1 µM). Note that only Tuni led to the accumulation of the ∼37 kD band of OSTM1. (B) HEK293T cells stably expressing OSTM1-Flag were treated with tunicamycin (5 µg/ml, 24 h) or PNGase-F (200 μg lysate protein/1,000 units, 4 h), and cell lysates were made. Cell lysates were also made from HEK293T cells stably expressing PDE3B. Indicated cell lysates were mixed and Flag or His antibody IP were performed. Note that tunicamycin and PNGase-F treatments led to de-glycosylated OSTM1, which showed stronger interactions with PDE3B than the glycosylated OSTM1 in untreated cell lysates. (C) OSTM1-Flag or OSTM1Δ31-Flag was stably expressed in the indicated BCL cell lines. Cells expressing OSTM1-Flag were treated with tunicamycin (5 µg/ml, 24 h). Cytosol/membrane fractionation was performed, and the indicated proteins were detected. Note that tunicamycin leads to de-glycosylation of OSTM1, and the signal peptide (SP)-less OSTM1Δ31 is predominantly localized in the cytosol. (D) PDE3B-His and indicated mutants of OSTM1Δ31-Flag were co-transfected into HEK293T cells. Reciprocal IP with His or Flag antibody was performed. Note that the CTD-less (Δ306-334) mutant lost the interaction with PDE3B.

Article Snippet: OSTM1-Flag expressing cell lysates underwent in-vitro PNGase-F treatment (NEB, P0704) under non-denaturing conditions following manufacturer’s protocol.

Techniques: Stable Transfection, Expressing, Membrane, Fractionation, Glycoproteomics, Transfection, Mutagenesis

Journal: bioRxiv

Article Title: OSTM1 is a ubiquitin E3 ligase that suppresses B-cell malignancy by activating the cAMP/PKA/CREB pathway

doi: 10.64898/2026.01.23.701155

Figure Lengend Snippet: (A) Wild-type full-length OSTM1-Flag or OSTM1Δ31-Flag were stably expressed in BCL lines. OSTM1Δ31, but not the full-length OSTM1, reduced PDE3B protein levels and cell growth. (B) BCL lines stably expressing OSTM1-Flag or OSTM1Δ31-Flag were treated with MG132 (10 µM for 8 h). OSTM1Δ31 expression led to decreased PDE3B levels, and was itself stabilized by MG132. (C) BCL lines stably expressing OSTM1-Flag were treated with tunicamycin. IB was performed and PDE3B level was quantified using ImageJ. Cell growth was determined by counting the number of cells. Tunicamycin led to de-glycosylation of OSTM1, decreased PDE3B stability, and resulted in more profound growth inhibition in OSTM1-expressing cells. (D) Cell lysates were prepared from HEK293T cells overexpressing PDE3B-His, overexpressing OSTM1-Flag treated with DMSO or tunicamycin, or overexpressing OSTM1Δ31-Flag. These lysates were used to perform in vitro ubiquitylation assays with the indicated combinations. OSTM1Δ31 and tunicamycin-treated OSTM1 lysates promoted PDE3B ubiquitylation. (E) Schematic showing the conserved zinc-coordinating Cys and His residues in the RING-like domain of OSTM1. The indicated Cys-to-Ala substitution mutants were generated in the OSTM1Δ31-Flag construct. (F) OSTM1-Flag, OSTM1Δ31-Flag and its C/A mutants were expressed in HEK293T cells. Flag IP was performed, followed by IB for ubiquitin to assess OSTM1 autoubiquitylation. OSTM1Δ31 C174A, C221A, and C275/279A mutants had markedly decreased autoubiquitylation. (G) OSTM1Δ31-Flag and its C/A mutants were expressed in human BCL lines. Endogenous PDE3B was detected by IB, and cell proliferation was measured. (H) CHX chase assay was performed in RPMI-8226 cells stably expressing OSTM1-Flag and OSTM1Δ31-Flag. PDE3B degradation kinetics was measured. (I) OSTM1Δ31-Flag or its C275/279A mutant was expressed in RPMI8226 cell line. PDE3B was pulled down and probed for ubiquitylation. OSTM1Δ31 enhanced PDE3B ubiquitylation, whereas the C275/279A mutant failed to do so. (J and K) CHX chase assay was performed in RPMI-8226 (J) and Z-138 (K) BCL lines stably expressing OSTM1Δ31-Flag or its C275/279A mutant. The half-lives of PDE3B and OSTM1Δ31 were determined by ImageJ densitometric analysis. Statistical significance was calculated using multiple t-tests, one per row. ***p<0.001; ****p<0.0001.

Article Snippet: OSTM1-Flag expressing cell lysates underwent in-vitro PNGase-F treatment (NEB, P0704) under non-denaturing conditions following manufacturer’s protocol.

Techniques: Stable Transfection, Expressing, Glycoproteomics, Inhibition, In Vitro, Generated, Construct, Ubiquitin Proteomics, Mutagenesis