pkm2 d78a4 rabbit mab  (Cell Signaling Technology Inc)

Journal: Inflammation

Article Title: PKM2 Promotes Glycolysis in Alveolar Macrophages and Induces Inflammation in Bronchopulmonary Dysplasia

doi: 10.1007/s10753-026-02476-9

Figure Lengend Snippet: Hyperoxia induces the increase in glycolysis levels in the lungs of BPD mice. a : Detect the lactate levels in the BALF of N7, H7, N14 and H14 groups. b : The mRNA expression level of PKM2 quantified by RT-qPCR. c : The activity of HK in the lung tissue of N7, H7, N14 and H14 groups. d : The protein levels of PKM2 in the lung tissue of N7, H7, N14 and H14 groups were detected by WB. e : Representative images of immunofluorescence after staining with anti-CD86 antibody (green) and anti-PKM2 antibody (red) at 14 days. Nuclei were stained by DAPI (blue). f : The relative fluorescence intensity of PKM2 in the lungs of each group at 14 days. Data are shown as means ± SD ( n = 3). * P < 0.05, ** P <0.01, *** P<0.001

Article Snippet: Primer antibodies used in this study: Anti-iNOS Mouse mAb (1:1000, Servicebio, China), PKM2 (D78A4) Rabbit mAb (1:1000, Cell Signaling Technology, USA) and Anti-beta actin Mouse mAb (1:1000, Servicebio, China).

Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Immunofluorescence, Staining, Fluorescence

Journal: Inflammation

Article Title: PKM2 Promotes Glycolysis in Alveolar Macrophages and Induces Inflammation in Bronchopulmonary Dysplasia

doi: 10.1007/s10753-026-02476-9

Figure Lengend Snippet: Hyperoxia induces the polarization of MH-S cells towards the M1 type and promotes inflammatory response. a : The mRNA expression of PKM2 in MH-S cells of normoxia and hyperoxia groups was detected by RT-qPCR. b : WB was used to detect the changes in iNOS and PKM2 levels in the normoxia and hyperoxia groups at the cellular level. c : The representative gating strategy of flow cytometry was used to identify CD86 + cells in MH-S cells. d : The mean fluorescence intensity and percentage of CD86 in normoxia and hyperoxia groups was analyzed. e: Concentrations of IL-1β and TNF-α in the cell supernatant of the normoxia and hyperoxia groups were measured by Elisa. Data are shown as means ± SD ( n = 3). * P < 0.05, ** P <0.01, *** P <0.001

Article Snippet: Primer antibodies used in this study: Anti-iNOS Mouse mAb (1:1000, Servicebio, China), PKM2 (D78A4) Rabbit mAb (1:1000, Cell Signaling Technology, USA) and Anti-beta actin Mouse mAb (1:1000, Servicebio, China).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Inflammation

Article Title: PKM2 Promotes Glycolysis in Alveolar Macrophages and Induces Inflammation in Bronchopulmonary Dysplasia

doi: 10.1007/s10753-026-02476-9

Figure Lengend Snippet: Graphical abstract. Hyperoxia induces the upregulation of PKM2 in alveolar macrophages, which affects the polarization and inflammatory level of M1 alveolar macrophages by mediating glycolysis, thereby promoting the development of BPD. Shikonin can weaken the polarization of M1 alveolar macrophages and pro-inflammatory response by inhibiting the activity of PKM2, and finally alleviate lung injury

Article Snippet: Primer antibodies used in this study: Anti-iNOS Mouse mAb (1:1000, Servicebio, China), PKM2 (D78A4) Rabbit mAb (1:1000, Cell Signaling Technology, USA) and Anti-beta actin Mouse mAb (1:1000, Servicebio, China).

Techniques: Activity Assay

Journal: Biomaterials

Article Title: Dual inhibition of oxidative phosphorylation and glycolysis using a hyaluronic acid nanoparticle NOX inhibitor enhanced response to radiotherapy in colorectal cancer.

doi: 10.1016/j.biomaterials.2025.123437

Figure Lengend Snippet: Fig. 3. Combination of HANP/GKT831 inhibited tumor progression and impacted metabolism with radiotherapy. HANP/GKT831 enhanced sensitivity to radiation and suppressed cell migration and invasion in the MC38 mouse tumor cells. A. Colony formation assay detected cell colonies resistant to 2 Gy of RT (n = 3). B. Transwell migration assay evaluated cell migration following the treatments. Cells were treated with 0.01 μM of conventional GKT831 or HANP/GKT831 con taining 0.01 μM of equivalent doses of GKT831. Quantification of 2D migration assay after 48 h in culture (n = 5). HANP/GKT831 reduced the protein levels of representative signal molecules in glycolysis, mitochondrial OXPHOS, and DNA repair pathways. C. The levels of Western blot analysis of NOX1, NOX4, glycolysis related (Hexokinase 2 and PKM2), and mitochondrial OXPHOS associated proteins (MT-ATP6 and MT-ND1). D. The protein levels in the cell cycle and DNA repair pathway, Cyclin D1, MSH6, and CHK1 proteins, were examined from the treated tumor lysates by Western blot analysis. β-actin served as the loading control. Similar results were obtained from at least 3 repeated studies. Student’s t-test: *p < 0.05 and ****p < 0.0001.

Article Snippet: After transferring to a polyvinylidene difluoride and blocking in 5 % of milk in tris-buffered saline (TBS) for 1 h, the blots were probed with rabbit anti-NOX1 antibody (MBS9609001, MYBioSource, San Diego, CA, USA), rabbit anti-NOX4 antibody (MBS820230, MYBioSource), anti-Hexokinase II rabbit monoclonal antibody (C64G5, Cell Signaling Technology), anti PKM2 XP rabbit monoclonal antibody (D78A4, Cell Signaling Technology), rabbit anti-MT-ATP6 antibody (70262, Cell Signaling Technology), rabbit anti-MT-ND1 antibody (NBP2-94462, NOVUS, MO, USA), anti-Cyclin D1 rabbit monoclonal antibody (2978, Cell Signaling Technology), rabbit anti-MSH6 antibody (MBS9626584, MYBioSource), rabbit anti-Phospho-Chk1 (Ser345) antibody (#2341, Cell Signaling Technology), hamster anti MCP-1 (CCL2) antibody (505911, Biolegend, San Diego, CA, USA), rabbit antiCXCL10 monoclonal antibody (701225, Invitrogen), anti-β-Actin mouse monoclonal antibody (A5316, Sigma-Aldrich).

Techniques: Migration, Colony Assay, Transwell Migration Assay, Western Blot, Control

Journal: bioRxiv

Article Title: The denitrosylase SCoR2 controls cardioprotective metabolic reprogramming

doi: 10.1101/2025.03.12.642752

Figure Lengend Snippet: (A) S-nitrosylated proteins in heart of SCoR2 +/+ (+/+) and SCoR2 -/- (–/–) mice post-I/R vs sham. Representative Coomassie-stained SDS/PAGE gel displaying SNO-proteins isolated by SNORAC using hearts of +/+ and –/– mice subjected to either sham operation or I/R (4hr reperfusion). Ascorbate was omitted from the SNORAC assay (-Asc) as a specificity control. ( B ): Three coordinated screens in +/+ vs. –/– mouse heart tissue, i.e. (1) SNORAC/MS (SCoR2-dependent S-nitrosoproteome 4 h after I/R, elevated >1.2-fold in –/– vs +/+) (Table S1; N=3), (2) SCoR2 co-IP interactome (Table S1; N=4), and (3) untargeted metabolomic screening in heart and plasma (Table S2; N=5 per condition), converge on the proteins BDH1 and PKM2 as SCoR2 substrate SNO-proteins in the heart which alter relevant cardioprotective metabolic pathways. ( C ) Full list of 31 overlapping proteins identified in both screens (1) and (2) i.e., the cardiac SCoR2-dependent S-nitrosoproteome and the cardiac SCoR2 interactome. Image created with Biorender.com.

Article Snippet: Antibodies employed in Western blotting included: rabbit polyclonal anti-AKR1A1/SCoR2 (15054-1-AP, Proteintech Group), rabbit monoclonal anti-PKM2 (D78A4, Cell Signaling), mouse monoclonal anti-p97 (10R-P104A, Fitzgerald), rabbit polyclonal anti-BDH1 (15417-1-AP, Proteintech Group).

Techniques: Staining, SDS Page, Isolation, Control, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: The denitrosylase SCoR2 controls cardioprotective metabolic reprogramming

doi: 10.1101/2025.03.12.642752

Figure Lengend Snippet: (A) Representative gel from SNORAC measuring SNO-BDH1 and Western blot measuring total BDH1, relative to p97 ATPase loading control, in mouse heart (4 hr reperfusion), quantified in . ( B ) SNO-BDH1 assessed by SNORAC in SCoR2 +/+ (+/+) and SCoR2 -/- (–/–) liver tissue (N=4 each) from mice (1hr reperfusion), quantified in . ( C ) Representative Western blot, quantified in , denoting a CHX pulse-chase assay, in which HEK293 cells transfected with V5-tagged BDH1 were treated with 100μg/mL CHX to block new protein synthesis, together with 100μM ECNO or vehicle, with samples collected at 6-24hr. Duplicates are shown by treatment condition. ( D,E ) Beta-hydroxybutyrate (β-HB) quantified as ion abundance in +/+ and –/– mouse heart and plasma by LC/MS-based untargeted metabolite profiling, N=5 mice per condition per genotype. ( F ) Sequences of BDH1, or protein product of closest homology, from selected species aligned with constraint-based multiple alignment tool ( COBALT ); red color shows differences from the H. sapiens BDH1 sequence. Cys 115 is indicated by black arrow. T. nigroviridis protein product ID is CAG04267. ( G,H ) Representative gel from SNORAC assessing SNO-BDH1 and SNO-PKM2 expression relative to SNO-p97 ATPase loading control (G) and from Western blot assessing BDH1, PKM2, and SCoR2 expression relative to p97 ATPase loading control (H) in human hearts without (N=10) and with (N=8-9) diagnosis of ischemic cardiomyopathy (ICM) (IRB # Pro00005621). Statistical significance in (D, E) determined by independent Student’s t-tests performed between genotypes at each time-point.

Article Snippet: Antibodies employed in Western blotting included: rabbit polyclonal anti-AKR1A1/SCoR2 (15054-1-AP, Proteintech Group), rabbit monoclonal anti-PKM2 (D78A4, Cell Signaling), mouse monoclonal anti-p97 (10R-P104A, Fitzgerald), rabbit polyclonal anti-BDH1 (15417-1-AP, Proteintech Group).

Techniques: Western Blot, Control, Pulse Chase, Transfection, Blocking Assay, Liquid Chromatography with Mass Spectroscopy, Sequencing, Expressing

Journal: bioRxiv

Article Title: The denitrosylase SCoR2 controls cardioprotective metabolic reprogramming

doi: 10.1101/2025.03.12.642752

Figure Lengend Snippet: (A) : Human heart samples (IRB# Pro00005621, N=9 with diagnosis of ischemic cardiomyopathy (ICM) and N=10 without known cardiac pathophysiology (healthy)) subjected to SNORAC measuring SNO-PKM2 relative to loading control (SNO-p97 ATPase). Representative SNORAC/Western blot gels shown in ,H. ( B-J ): Metabolites quantified by ion abundance in SCoR2 +/+ (+/+) and SCoR2 -/- (–/–) mouse heart and plasma by LC/MS-based untargeted metabolite profiling; N=5 each condition. (B) Heart lactate. (C-K) Metabolites organized by relationship to the PPP, as inputs (C,D), products (E-H), or polyol compounds that are categorized as downstream end products of the PPP (I,J). NADPH measured in heart lysate after 2 hr reperfusion, erythrose 4-phosphate measured after 4 hr reperfusion. ( K ): Recombinant SCoR2 activity quantified via NADPH consumption by spectrophotometer in the presence of canonical substrate (100μM SNO-CoA) or carbohydrates (1mM); [SCoR2] = 186nM, [NADPH] = 100μM. Results presented as specific activity of SCoR2 (μM substrate consumed/min/mg protein). Assay performed in triplicate. ( L ): Summary model showing SCoR2-mediated regulation of carbohydrate metabolism, including PPP and polyol compounds, to generate NADPH and phosphocreatine in the mouse heart. Green arrows indicate pathway upregulation in the absence of SCoR2 and red arrows indicate downregulation. Blue boxes indicate metabolic pathways, tan boxes indicate metabolites, and pink boxes indicate metabolites of particular significance. Thick black arrows indicate directions of SCoR2-regulated metabolic changes. Image created with Biorender.com. Statistical significance in (A) determined by Student’s t-test; (B-H,J) determined by multiple independent Student’s t-tests performed between genotypes in each condition; (I) determined by two-tailed Mann-Whitney test; (K) determined by two-tailed Mann-Whitney test between SNO-CoA condition and each carbohydrate condition (N = 3-8 independent replicates per condition). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Antibodies employed in Western blotting included: rabbit polyclonal anti-AKR1A1/SCoR2 (15054-1-AP, Proteintech Group), rabbit monoclonal anti-PKM2 (D78A4, Cell Signaling), mouse monoclonal anti-p97 (10R-P104A, Fitzgerald), rabbit polyclonal anti-BDH1 (15417-1-AP, Proteintech Group).

Techniques: Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Recombinant, Activity Assay, Spectrophotometry, Two Tailed Test, MANN-WHITNEY