Journal: Frontiers in Immunology

Article Title: Peptidoglycan Recognition Protein 4 Limits Bacterial Clearance and Inflammation in Lungs by Control of the Gut Microbiota

doi: 10.3389/fimmu.2019.02106

Figure Lengend Snippet: Differential regulation of Pglyrp4 and candidate gene expression in primary cells. The Pglyrp4 expression was measured by qPCR in in vitro -stimulated ( S. pneumoniae D39, MOI 1, 6 h) vs. unstimulated WT primary (A) alveolar epithelial cells (AECs), alveolar macrophages (AMΦs), and bone marrow-derived neutrophils (PMNs). (B) The expression of the tight junction genes Cldn18, F11r , and Cdh1 in PGLYRP4KO vs. WT AECs as well as (C) complement C3 , (D) Ifng , and (E) Ifngr1 in PGLYRP4KO vs. WT AECs, AMΦs, and PMNs was analyzed after infection ( S. pneumoniae D39, MOI 1, 6 h). Relative expression was calculated by the ΔΔ C T method with Gapdh as the housekeeping gene and uninfected WT cells as the control. Values are expressed as means + SEMs ( n = 3–5). The dotted line represents the level of uninfected WT cells. Statistical analysis: Student's t -test: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns p ≥ 0.1 vs. untreated control.

Article Snippet: Total RNA (1 μg) was transcribed into cDNA (High-Capacity cDNA Reverse Transcription Kit), pre-amplified (TaqMan PreAmp Master Mix Kit), and used for qPCR (TaqMan Gene Expression Master Mix and TaqMan Assay Sequence Numbers: Gapdh Mm99999915_g1, Pglyrp4 Mm01220032_m1, Cldn18 Mm00517321_m1, F11r Mm00554113_m1, Cdh1 Mm01247357_m1, C3 Mm01232779_m1, Ifng Mm01168134_m1, Ifngr 1 Mm00599890_m1; all Life Technologies GmbH, Darmstadt, Germany).

Techniques: Gene Expression, Expressing, In Vitro, Derivative Assay, Infection, Control

Journal: International Journal of Molecular Sciences

Article Title: UBC9-Mediated SUMO Pathway Drives Prohibitin-1 Nuclear Accumulation and PITX1 Repression in Primary Osteoarthritis

doi: 10.3390/ijms26136281

Figure Lengend Snippet: PHB1 can bind SUMO1 proteins via a SIM (SUMO-interacting module), which is crucial for its nuclear localization. ( a ) Diagram represents wild-type PHB1 protein structure and various PHB1 constructs: wild-type PHB1 (WT PHB1), a mutant where the nuclear signal of export was deleted (PHB1-∆NES) or was replaced by a nuclear localization signal (PHB1-NLS), and a mutant where a putative SUMO-interacting motif (SIM) was deleted (PHB1-∆SIM). All the constructs have a triple Flag-tag at the N-terminal. ( b ) Co-immunoprecipitation assays with anti-c-Myc antibodies demonstrate that PHB1 interacts with Myc-tagged SUMO1 through the SIM (upper panel). The lower panel indicates the level of Myc-tagged SUMO1 protein in total cell extracts (X-T). ( c ) The nuclear accumulation of PHB1 is dependent on its SIM. C28/I2, a human chondrocyte cell line, were infected with either flag-tagged wild type (PHB1), (PHB1_NLS), or (PHB1_∆SIM) constructs or empty vector, to produce stable cell lines. The nuclear extract (X-N) and the cytoplasmic extract (X-C) proteins were isolated and analyzed by Western immunoblotting to detect the subcellular presence of flag-tagged PHB1. Anti-GAPDH was used as a cytoplasmic loading control, and anti-Lamin was used as a nuclear loading control. Note the significantly reduced nuclear presence of PHB1-ΔSIM compared to WT PHB1 and PHB1-NLS.

Article Snippet: For Western blotting, we used antibodies against the following: GAPDH (V-18: sc-20357, dilution [1:5000]) and PHB1 (H-80: sc-28259, dilution [1:500]) (both from Santa Cruz Biotechnology Inc., Dallas, TX, USA); lamin A/C (#2032; dilution [1:2000]), SUMO-1 (A21C7; dilution [1: 2000]), and SUMO-2/3 (18H8; dilution [1:2000]) (all from Cell Signaling Technology Inc., Danvers, MA, USA); FLAG (Sigma M2 F3165; dilution [1:5000]) and RanGap1 (33-0800; dilution [1: 2000]) (both from Zymed Technologies—Thermo Fisher Scientific Inc., Waltham, MA, USA); and GST (M1; dilution [1: 2000]) (from EMD Millipore, Burlington, MA, USA).

Techniques: Construct, Mutagenesis, FLAG-tag, Immunoprecipitation, Infection, Plasmid Preparation, Stable Transfection, Isolation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: UBC9-Mediated SUMO Pathway Drives Prohibitin-1 Nuclear Accumulation and PITX1 Repression in Primary Osteoarthritis

doi: 10.3390/ijms26136281

Figure Lengend Snippet: The UBC9-mediated SUMO pathway stabilizes PHB1 and promotes its nuclear accumulation in U2OS cells. ( a ) Co-expression of UBC9 and SUMO isoforms enhances PHB1 protein levels. U2OS cells were transfected with the pLPC-3xFlag-PHB1 alone or co-transfected with different components of the SUMOylation pathway (UBC9; UBC9 + Sumo1; UBC9 + Sumo2; UBC9 + Sumo3). Total cell lysates were analyzed by Western blotting using an anti-Flag antibody to detect Flag-PHB1 protein levels. ( b ) The nuclear accumulation of PHB1 is dependent on its SIM in the presence of UBC9 and SUMO-1. U2OS cells were transfected with Flag-tagged PHB1, PHB1-NLS, or PHB1-ΔSIM constructs, in the presence or absence of co-transfected Myc-SUMO-1 and UBC9. The nuclear proteins (=X-N), as well as total proteins (X-T), were isolated from cells transfected with pLPC-3xFlag-PHB1, PHB1-NLS, or PHB1-∆SIM in the presence or absence of myc-SUMO1 and UBC9. Anti-GAPDH was used as a cytoplasmic loading control, and anti-Lamin was used as a nuclear loading control, demonstrating successful cell fractionation. ( c ) U2OS cells were transfected with UBC9 alone or co-transfected with pCMV4-myc-SUMO1, HA-SUMO2, and myc-SUMO3 or with the empty vector. Total (T), cytoplasmic (C), and nuclear (N) protein extracts were isolated. Western blot analysis using an anti-PHB1 antibody reveals changes in endogenous PHB1 subcellular localization. Anti-GAPDH and Anti-Lamin A/C were used as loading controls for cytoplasmic and nuclear fractions, respectively.

Article Snippet: For Western blotting, we used antibodies against the following: GAPDH (V-18: sc-20357, dilution [1:5000]) and PHB1 (H-80: sc-28259, dilution [1:500]) (both from Santa Cruz Biotechnology Inc., Dallas, TX, USA); lamin A/C (#2032; dilution [1:2000]), SUMO-1 (A21C7; dilution [1: 2000]), and SUMO-2/3 (18H8; dilution [1:2000]) (all from Cell Signaling Technology Inc., Danvers, MA, USA); FLAG (Sigma M2 F3165; dilution [1:5000]) and RanGap1 (33-0800; dilution [1: 2000]) (both from Zymed Technologies—Thermo Fisher Scientific Inc., Waltham, MA, USA); and GST (M1; dilution [1: 2000]) (from EMD Millipore, Burlington, MA, USA).

Techniques: Expressing, Transfection, Western Blot, Construct, Isolation, Control, Cell Fractionation, Plasmid Preparation