ampk2 protein  (Proteintech)

Journal: bioRxiv

Article Title: Smoothened turnover regulated by Hedgehog signaling in Drosophila

doi: 10.64898/2026.01.08.698469

Figure Lengend Snippet: (A) Schematic of frontal depiction of the wing pouch of the wing disc with the A (gray) and P (green) compartments indicated by gray and green, respectively; boxes indicate positions of (F-K) images. (B) White dotted lines delineate wing pouch and compartment border of disc expressing BAC encoded Hh:GFP. (C) Sagittal section of the wing disc expressing BAC encoded Hh:GFP (α-GFP antibody staining). Septate junction stained with α-DLG (red) marks septate junction; phalloidin marks adherens junction (blue). (D) Graph of Hh:GFP fluorescence in A and P compartments. (E) Schematic of sagittal wing pouch section with apical and basolateral compartments, Hh:GFP (green) and Rab7 (red) indicated. (F-G”) Frontal views of region of the A compartment of wing discs with BAC-encoded Hh:GFP detected with α-GFP antibody (green) and stained with α-Rab7 antibody (red) and Phalloidin (blue). (H,H’) Sagittal section showing total Hh signal (H) and Hh not colocalized with Rab7. White and yellow arrows indicate peripodial membrane and apical compartment, respectively. (I-K’) Same as (F-H’) for P compartment. (K) Graph showing the proportion of BAC encoded Hh:GFP signal that overlaps with Rab7 in apical/basolateral optical sections.

Article Snippet: The following antibodies were used: mouse α-GFP (Roche), rabbit α-RFP (Rockland), mouse α-Ptc (DSHB, Apa1), mouse α-En (DSHB, 4D9), DAPI, Phalloidin, mouse α-Dlg1, (DSHB, 4F3), α-Rab7 , α-Smo (DSHB, 20C6), Alexa633 conjugated-Phalloidin (Invitrogen), DAPI (Invitrogen), AbN ( ).

Techniques: Expressing, Staining, Fluorescence, Membrane

Journal: bioRxiv

Article Title: Smoothened turnover regulated by Hedgehog signaling in Drosophila

doi: 10.64898/2026.01.08.698469

Figure Lengend Snippet: (A-E) Frontal views of unfixed wing discs, optical section close to the apical surface; left, anterior; right, posterior. (A,B) Wing discs expressing BAC-encoded GFP:Cherry:Smo without (A) and with ammonium chloride (B); GFP channel (green), RFP channel (red). (C) Wing disc expressing BAC-encoded GFP:Smo (green) and Rbcn3A RNAi (expressed during L3 at 29°C) in the dorsal region (below white dashed line) using ( ap-Gal4/tub-Gal80 ts ; GFP:Smo/Rbcn3A-RNAi ). (D,E) Unfixed wing discs expressing Cherry:Smo (red) and Rab5:YFP (green) (D) or Cherry:Smo (red) and Rab7:YFP (green). (F) Amount of Cherry:Smo/Rab7 double positive and Cherry:Smo /Rab5 double positive puncta.

Article Snippet: The following antibodies were used: mouse α-GFP (Roche), rabbit α-RFP (Rockland), mouse α-Ptc (DSHB, Apa1), mouse α-En (DSHB, 4D9), DAPI, Phalloidin, mouse α-Dlg1, (DSHB, 4F3), α-Rab7 , α-Smo (DSHB, 20C6), Alexa633 conjugated-Phalloidin (Invitrogen), DAPI (Invitrogen), AbN ( ).

Techniques: Expressing

Journal: bioRxiv

Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

doi: 10.64898/2026.01.27.700981

Figure Lengend Snippet: (a) Schematic of immunoassay for cytokine detection. (b) Initial LOD optimization for the IFN-γ assay. (c-e) IFN-γ (n=2), IL-2 (n=5), TNF-α (n=2) dilution curves using recombinant cytokines (f-h) Correlation plots of ELISA absorbance values at 450 nm versus silver darkness values for IFN-γ (f), TNF-α (g), and IL-2 (h). All fit lines are four-parameter logistic sigmoidal curves.

Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

doi: 10.64898/2026.01.27.700981

Figure Lengend Snippet: (a) Schematic demonstrating the stimulation groups and controls. Dilution curves for IFN-γ (b-c), IL-2 (d-e), and TNF-α (f-g) in pooled LTB+ and LTB-clinical supernatant samples. Clinical samples consisted of NIL (negative control), Ag1, Ag2, and Mitogen (positive control) groups (n=2).

Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

Techniques: Negative Control, Positive Control

Journal: bioRxiv

Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

doi: 10.64898/2026.01.27.700981

Figure Lengend Snippet: Individual participant silver darkness values for IFN-γ (a), IL-2 (b), and TNF-α (c) detection from NIL, Ag1, Ag2, and Mitogen samples of IGRA+ and IGRA-patients using a sample dilution of 1:10. Average of n=2 repeats was plotted for each sample.

Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

Techniques:

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: In general, non-receptor interacting loops were deleted from the WT-IL7 sequence and loops connecting the adjacent helices were modeled using Rosetta Loop Remodeler and Rosetta fix backbone design function. The sequence of the designed model was extracted and submitted to AlphaFold (monomer and multimer mode for structure and protein-receptor binding prediction respectively) as a preliminary validation of the Rosetta-remodeled protein. Iterations of the bad models (models that do not fold into the expected structure or models that did not predict to bind to the receptors) back to the design stage were performed. Models that passed the AlphaFold validation proceeded to subsequent in vitro assay using yeast display system and flow cytometry to determine their relative binding affinity to IL-7 receptors in comparison to WT-IL7.

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques: Sequencing, Binding Assay, Biomarker Discovery, In Vitro, Flow Cytometry, Comparison

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: Blueprint of the WT-IL7 was shown on the left of the figure. The connectivity of the functioning helixes was connected in a manner that requires extremely long protein loops by design (i.e. helices were not connected to the closest adjacent helixes but to the opposite helix). Loops that were not interacted with the IL-7 receptors were deleted and the helixes were reconnected in a clockwise manner via new protein linkers connecting to the adjacent helixes. The blueprint of the redesigned protein was shown at the right side of the figure. Protein structures are colored as rainbow (from N-to-C terminus with the order of Blue-Green-Yellow-Red).

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques:

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: ( A ) AlphaFold validation of the first loop design version of Neo-7 (Neo-7 LDv1) using the default (left) and single sequence mode (right). ( B ) AlphaFold validation of the second loop design version of Neo-7 (left; Neo-7 LDv2) and Neo-7 LDv2 with mutations (right) favored by Rosetta fix backbone design. ( C ) Crystal structure of human IL-7 in complexation to human IL-7 receptor alpha (PDB ID = 3DI2). ( D ) Superimposition of Neo-7 structures (with or without additional disulfide bridge) predicted by AlphaFold. ( E ) Yeast display and flow cytometry validation of IL-7/Neo-7 bindings towards the IL-7 receptors. The yeast-displayed protein (different redesigned IL-7s) carries a HA-tag while the recombinant IL-7 receptors carry either a HIS tag (IL-7 receptor alpha) or a FC-tag (common-IL-2 family receptor gamma; for detection of IL-2Rγ binding, yeast cells were first incubated with recombinant IL-7Rα, washed, and subsequently incubated with IL-2Rγ.) The signal intensity of the X-axis (conferred by the binding of anti-HA mab) correlates with the expression level of the displayed protein while the signal intensity of the Y-axis (conferred by the binding of the anti-HIS/anti-FC mAb to the recombinant receptors bound to the displayed proteins) correlates with the binding affinity of the displayed proteins towards the IL-7 receptors.

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques: Biomarker Discovery, Sequencing, Flow Cytometry, Recombinant, Binding Assay, Incubation, Expressing

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: ( A ) Inspection of structural and binding interactions of residue mutations Q6P and T45I on Neo-7 towards the murine IL-7R alpha. ( B ) Yeast display and flow cytometry validation of the binding ability of IL-7/Neo-7 variants toward the IL-7 receptors.

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques: Binding Assay, Residue, Flow Cytometry, Biomarker Discovery

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: FPLC profile of E. coli expressed ( A ) WT-IL7 ( B ) refolded WT-IL7 ( C ) Neo-7-Q6P and ( D ) Neo-7-Q6P-T45I. Percentage of purity is calculated from the SEC-FPLC peak profile via Cytiva Unicorn 7 software after affinity chromatography purification. SPR (Biacore) characterization of the binding kinetics of ( E ) Neo-7-Q6P ( F ) Neo-7-Q6P-T45I and ( G ) WT-IL7 towards murine IL-7R alpha. ( H ) 2E8 proliferation assay to investigate the biological activity of the IL-7/Neo-7s expressed by E. coli . Error bars represent standard deviation (n=3).

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques: Software, Affinity Chromatography, Purification, Binding Assay, Proliferation Assay, Activity Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Identification of KHS-101 as a Transcription Factor EB Activator to Promote α-Synuclein Degradation

doi: 10.3390/ijms27020905

Figure Lengend Snippet: KHS-101 increases α-synuclein (A53T) degradation via autophagic–lysosomal system. ( A , B ) Representative immunoblot ( A ) and quantification ( B ) of α-synuclein levels. SH-SY5Y cells were treated with KHS-101 (1 μM, 2 μM, 4 μM, 6 μM) and doxycycline (1 μg/mL) for 48 h. ( C , D ) Representative immunofluorescence images ( C ) and quantification ( D ) of α-synuclein levels in SH-SY5Y cells obtained after 48 h treatment with KHS-101 (6 μM) and doxycycline (1 μg/mL). Scale bars represent 10 μm. ( E , F ) α-synuclein degradation by KHS-101 was abolished in the presence of CQ. SH-SY5Y cells were treated with KHS-101 (6 μM) and doxycycline (1 μg/mL) for 48 h with or without CQ (5 μM). All data are expressed as mean ± SEM (** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: The cDNA sequences of TFEB and α-synuclein (A53T) were purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Immunofluorescence