leupeptin  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: GAPDH is modified with carboxyethylation at Cys 247 (A) Mass spectrometry analysis of GAPDH peptide (234–260) with carboxyethylation and 3-HPA (5 mM) incubated with the GAPDH peptide (234–260) at 37 °C for 4 h. (B) SPR analysis of the affinity of the anti-ceC247 antibody for the carboxyethylation modified GAPDH peptide (234–260) and unmodified GAPDH peptide (234–260). (C) ELISA-based binding curve of anti-ceC247 antibody to modified GAPDH peptide (GAPDH ce (234–260)) and unmodified GAPDH peptide (234–260). Data are the means ± SD and n = 3 per group. Statistical significance was determined using two-way ANOVA followed by ∗∗p < 0.01. (D) Chemical structures of cysteine carboxyethylation and cysteine lactylation. (E) Unmodified GAPDH peptide (234–260), carboxyethylated peptide (GAPDH ce (234–260)), and lactylated peptide (GAPDH lac (234–260)) were tested with the anti-ceC247 antibody in dot blot assays. (F) Immunoblots of lysates from 293 T cells overexpressing GAPDH, which were treated with 5 mM 3-HPA and 5 μM MG132. The blots were probed with the anti-ceC247 antibody. (G) 3-HPA incubated with the GAPDH peptide (234–260) at 37 °C for 4 h. An anti-ceC247 antibody and the anti-wtC247 antibody were used in dot blot assays.

Article Snippet: To determine the degradation pathway, cells were treated with 5 μM MG132 (MCE), 100 μM Leupeptin (MCE), 5 nM bafilomycin A1 (MCE), or 50 μM chloroquine (MCE) for 24 h. The stability of GAPDH was analyzed by immunoblotting as described above.

Techniques: Modification, Mass Spectrometry, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Dot Blot, Western Blot

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: 3-HPA-induced carboxyethylation of GAPDH promotes its degradation through the ubiquitin-proteasome pathway (A) Chemical structure of carboxyethylated cysteine (left) and structures of aspartic acid (D), glutamic acid (E), methionine (M), and cysteine (C). (B) Immunoblot of GAPDH after transient transfection of flag-tagged GAPDH(C), GAPDH(D), GAPDH(M), GAPDH(E) plasmid in 293 T cells at 24 h, 36 h, and 48 h. (C) Immunoblot of GAPDH after CHX treatment. The GAPDH antibody was used to compare the degradation rates of GAPDH(M), GAPDH(E), GAPDH(D), and GAPDH(C). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05; ns, not significant. (D) Immunoblot and quantitative analysis of GAPDH ce after CHX treatment. The anti-ceC247 antibody and anti-GAPDH antibody were used to compare the degradation rates of carboxyethylated GAPDH. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01; ns, not significant. (E) Immunoblot and quantitative analysis of GAPDH ce after 3-HPA treatment. The anti-ceC247 antibody and anti-GAPDH antibody were used to compare the content of carboxyethylated GAPDH and total GAPDH. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05; ∗∗p < 0.01; ns, not significant. (F) Immunoblot and quantitative analysis of GAPDH ce in 293 T cells treated with 3-HPA (5 mM) combined with proteasomal inhibitor MG132, autophagic inhibitor Chloroquine, or lysosomal inhibitor Bafilomycin A1. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01; ∗∗∗∗p < 0.0001; ns, not significant. (G) Immunoprecipitation of GAPDH or GAPDH ce followed by immunoblotting for Myc in 293 T cells transfected with Myc-Ub. Cells were treated with 3-HPA (5 mM) and MG132 (5 μM) for 24 h. (H) Immunoprecipitation of GAPDH or GAPDH ce followed by immunoblotting for Myc in 293 T cells transfected with Myc-Ub mutants (K6O, K11O, K27O, K29O, K33O, K48O, K63O). Cells were treated with 3-HPA (5 mM) for 24 h.

Article Snippet: To determine the degradation pathway, cells were treated with 5 μM MG132 (MCE), 100 μM Leupeptin (MCE), 5 nM bafilomycin A1 (MCE), or 50 μM chloroquine (MCE) for 24 h. The stability of GAPDH was analyzed by immunoblotting as described above.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Translational Lung Cancer Research

Article Title: TLR7 induces autophagy in non-small cell lung cancer tumor cells and influences anti-tumors responses in patients

doi: 10.21037/tlcr-2025-aw-1173

Figure Lengend Snippet: Study the impact of TLR7 on the autophagy level in lung tumor cells. (A) Example of images of A549 stably transfected with the GFP-LC3 construct (A549 GFP-LC3) treated or not (NT) with an autophagy inducer, the rapamycin (Rapa., 1 µM) or by synthetic TLR7 agonists, CL264 (1 mM) and loxoribin (Loxo., 1 mM) for 6h. The graph represents the quantification of the GFP-LC3 dots per cell in each condition at 6 h post-treatment. The experiment was performed six times. (B) The same experiment as in A was performed with different kinetics: 6, 24, and 48h post-treatments. The graph represents the evolution of the GFP-LC3 dots per cell in each condition over time. The experiment was performed six times. (C) Example of images of A549 GFP-LC3 treated or not (NT) with either inhibitors of the autophagy maturation, Leupeptin and E64d (L + E, 1 ug/mL each), an autophagy inducer (Rapa., 1 µM) or synthetic TLR7 agonists (CL264 and Loxo., 1 mM) for 24 h. The graph represents the quantification of the GFP-LC3 dots per cell in each condition. The experiment was performed three times. (D) Example of images of A549 GFP-LC3 treated or not (NT) with either an inhibitor of the autophagy maturation, the Baf. (100 nM), an autophagy inducer (Rapa., 1 µM) or synthetic TLR7 agonists (Loxo., 1 mM) for 24 h. Lysosomes were marked in red by the addition of lysotracker 90 minutes before the end of the experiment. The graph represents the study of pixel colocalization (green versus red puncta) using the coloc2 script from the ImageJ software. The experiment was performed three times. (E) A549 cells were transfected with a plasmid encoding a dual-labeled LC3 probe, RFP-GFP-LC3. Cells were again treated with Rapa. or with Loxo. (1 mM) for 24 h. Representative images are shown and the number of total autophagic vacuoles (GFP + RFP + + GFP − RFP + ), autophagosomes (GFP + RFP + ), and autolysosomes (GFP − RFP + ) were enumerate. The inset permits visualization of yellow autophagosome (yellow arrow) and red autolysosomes (red arrow). The experiment was performed three times. (F) At the left: measurement of the proportion of lung tumor cells LC3 positive versus negative for both TLR7 low (proportion of lung tumor cells expressing TLR7 <78%) versus TLR7 high (proportion of lung tumor cells expressing TLR7 >78%) NSCLC patients of the cohort 1. In the right: implementation of an autophagy score (score 1, proportion of lung tumor cells LC3 pos <25%; 2, 25%< x <50%; 3, 50%< x <75% and 4, x >75%). Evaluation of the TLR7 expression level in function of the autophagy score. (G) The same analysis as in E was carried out on the NSCLC patients from cohort 2, those treated by neoadjuvant chemotherapies. Student’s t -test, *, P<0.05. Baf., bafilomycin; GFP, green fluorescent protein; LC3, light chain 3; Loxo., loxoribin; NSCLC, non-small cell lung cancer; Rapa., rapamycin; RFP, red fluorescent protein; TLR7, toll-like receptor 7.

Article Snippet: A1 (100 nM, Sigma) or a combination of leupeptin hemisulfate (1 μg/mL, Invitrogen) and E64D (1 μg/mL, Invitrogen).

Techniques: Stable Transfection, Transfection, Construct, Software, Plasmid Preparation, Labeling, Expressing

Journal: BioMedical Engineering OnLine

Article Title: Synergistic enhancement of peripheral nerve regeneration using electrospun polylactic acid conduits with leupeptin and methylprednisolone in rats

doi: 10.1186/s12938-026-01565-y

Figure Lengend Snippet: Chart 1–8 The difference of SFI among all groups began to be statistically on the timepoint of 10 weeks after surgery, and then the variation became more obvious. The level of the combined intervention group was always at the top, followed by the leupeptin group and the glucocorticoid group.

Article Snippet: Considering the recommended concentration in the protocol of purchased leupeptin (MCE, USA) and the local application of methylprednisolone in previous studies [ ], the final 10 mm nerve gap was filled with 10 μL of saline (saline group), 0.5 mg/mL leupeptin (leupeptin group), 30 mg/mL methylprednisolone (glucocorticoid group), and 0.5 mg/mL leupeptin + 30 mg/mL methylprednisolone (combined intervention group).

Techniques:

Journal: BioMedical Engineering OnLine

Article Title: Synergistic enhancement of peripheral nerve regeneration using electrospun polylactic acid conduits with leupeptin and methylprednisolone in rats

doi: 10.1186/s12938-026-01565-y

Figure Lengend Snippet: Mean fluorescence intensity reflects the protein expression levels. As illustrated in the figure, collagen I and collagen III expression levels were consistently lowest in the combined intervention group, highest in the saline group, and intermediate in the leupeptin group and the glucocorticoid group. Furthermore, the glucocorticoid group exhibited lower expression levels of collagen I and collagen III compared to the leupeptin group.

Article Snippet: Considering the recommended concentration in the protocol of purchased leupeptin (MCE, USA) and the local application of methylprednisolone in previous studies [ ], the final 10 mm nerve gap was filled with 10 μL of saline (saline group), 0.5 mg/mL leupeptin (leupeptin group), 30 mg/mL methylprednisolone (glucocorticoid group), and 0.5 mg/mL leupeptin + 30 mg/mL methylprednisolone (combined intervention group).

Techniques: Fluorescence, Expressing, Saline

Journal: BioMedical Engineering OnLine

Article Title: Synergistic enhancement of peripheral nerve regeneration using electrospun polylactic acid conduits with leupeptin and methylprednisolone in rats

doi: 10.1186/s12938-026-01565-y

Figure Lengend Snippet: Masson staining of the tissue in the middle of the electrospun nerve conduit at 20× magnification. Nerve tissue is dyed red and collagen fibers are dyed blue. A In the saline group, the nerve tissue failed to pass through the nerve conduit. The nerve tissue was full of collagen fibers. B In the leupeptin group, the nerve tissue filled the nerve conduit, but it was narrower than that in the glucocorticoid group and the combined intervention group, and the collagen fibers in the nerve tissue were significantly reduced. C In the glucocorticoid group, the nerve tissue filled the nerve conduit, and the collagen fibers in the nerve tissue were minimal. D In the combined intervention group, the nerve tissue was filled with nerve conduit, the nerve tissue was the widest, and the collagen fibers in the nerve tissue were also less than that in the saline group. The black arrow shows the wall of the nerve conduit. The yellow arrow shows the hollow of the nerve conduit. All pictures were annotated with a 100-μm scale bar.

Article Snippet: Considering the recommended concentration in the protocol of purchased leupeptin (MCE, USA) and the local application of methylprednisolone in previous studies [ ], the final 10 mm nerve gap was filled with 10 μL of saline (saline group), 0.5 mg/mL leupeptin (leupeptin group), 30 mg/mL methylprednisolone (glucocorticoid group), and 0.5 mg/mL leupeptin + 30 mg/mL methylprednisolone (combined intervention group).

Techniques: Staining, Saline

Journal: BioMedical Engineering OnLine

Article Title: Synergistic enhancement of peripheral nerve regeneration using electrospun polylactic acid conduits with leupeptin and methylprednisolone in rats

doi: 10.1186/s12938-026-01565-y

Figure Lengend Snippet: Saline group exhibited the highest collagen fiber area ratio, while the glucocorticoid group showed the lowest one among all groups. The combined intervention group demonstrated a higher collagen fiber area ratio compared to the glucocorticoid group, and the leupeptin group displayed a slightly elevated proportion relative to the combined intervention group. The reliable antifibrotic effect of glucocorticoids may account for the lowest degree of fibrosis observed in the glucocorticoid group. Although fibrosis was slightly higher in the combined intervention group compared to the glucocorticoid group, the amount of nerve fiber tissue within the conduits and the extent of nerve regeneration were significantly higher in the combined intervention group.

Article Snippet: Considering the recommended concentration in the protocol of purchased leupeptin (MCE, USA) and the local application of methylprednisolone in previous studies [ ], the final 10 mm nerve gap was filled with 10 μL of saline (saline group), 0.5 mg/mL leupeptin (leupeptin group), 30 mg/mL methylprednisolone (glucocorticoid group), and 0.5 mg/mL leupeptin + 30 mg/mL methylprednisolone (combined intervention group).

Techniques: Saline