Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 2. Effects of integrin β4 knockdown in VECs on VSMC contraction. (A) VECs and VSMCs were co-cultured in the micropore membrane insert well co-culture system for 24 h. Then, the levels of integrin β4 in the VECs were examined using the immunofluorescence assay. The bar graph shows the relative intensity of integrin β4 in the single-cultured and co-cultured VECs (bP<0.05 vs single-cultured VECs, n=3). (B) SiRNA-mediated down-regulation of integrin β4 in VECs. The levels of integrin β4 were determined by western blotting 48 h after the start of the siRNA treatment. In the scramble control group (scramble ctrl), cells were transfected with scramble control siRNA (cP<0.01 vs scramble ctrl, n=3). (C) VECs were treated with 20 nmol/L integrin β4-specific siRNA or with scramble siRNA for 48 h. After the addition of SPC, the effects of the integrin β4 knockdown on the contraction of the VSMCs in the micropore membrane insert well system were observed under a phase contrast microscope (×200) and analyzed by the collagen contractility assay. (D) The bar graph shows the relative contractility following SPC treatment, which is represented as the ratio of the gel area to that of the untreated control (cP<0.01 vs SPC-stimulated scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Membrane, Co-Culture Assay, Immunofluorescence, Western Blot, Control, Transfection, Microscopy

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 3. Effects of Fyn knockdown in VECs on VSMC contraction. (A) VECs and VSMCs were co-cultured in the micropore membrane insert well co- culture system for 24 h. Then, the levels of Fyn in the VECs were examined using the immunofluorescence assay. The bar shows the relative intensity of Fyn in single-cultured and co-cultured VECs (bP<0.05 vs single-cultured VECs, n=3). (B) SiRNA-mediated down-regulation of Fyn in VECs. The value of Fyn was determined by western blotting 48 h after the start of the siRNA treatment. In the scramble control group (scramble ctrl), the cells were transfected with scramble control siRNA (cP<0.01 vs scramble ctrl, n=3). (C) VECs were treated with 20 nmol/L Fyn-specific siRNA or with scramble siRNA for 48 h. After the SPC was added, the effects of the Fyn knockdown on the contraction of the VSMCs in the micropore membrane insert well system were observed under a phase contrast microscope (×200) and analyzed using the collagen contractility assay. (D) The bar graph shows the relative contractility following SPC treatment, which is represented as a ratio of the gel area to that of the untreated control (cP<0.01 vs SPC-stimulated scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Membrane, Co-Culture Assay, Immunofluorescence, Western Blot, Control, Transfection, Microscopy

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 4. Effects of integrin β4 knockdown on the changes in Fyn levels in VECs. (A) VECs were treated with 10–40 nmol/L integrin β4 siRNA or scramble siRNA for 48 h. The levels of integrin β4 in the VECs were determined by the immunofluorescence assay, and the bar graph shows the relative fluorescent intensity of integrin β4 per cell as determined by laser scanning confocal microscopy (bP<0.05 vs scramble ctrl, n=3). (B) After 10–40 nmol/L integrin β4 siRNA or scramble siRNA were treated for 48 h, the Fyn levels in the VECs were assessed using western blotting (cP<0.01 vs scramble ctrl, n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Immunofluorescence, Confocal Microscopy, Western Blot

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 5. Effects of integrin β4 knockdown on NO production in VECs. (A) VECs were cultured under normal conditions or with siRNA for 48 h and stimulated with SPC for 0, 3, 15, or 30 min, and the supernatant was used for the NO level assay using the NO Detection Kit. In the control group (ctrl), the cells were cultured in M199 medium with 0.3% (v/v) ethanol instead of SPC. In the scramble control group (scramble ctrl), cells were transfected with scramble control siRNA in the presence of SPC. The bar graph shows the changes in NO levels in VECs that had been treated with SPC (bP<0.05 vs ctrl at 3 min, eP<0.05 vs ctrl at 15 min, n=5) or with siRNA in the presence of SPC (hP<0.05 vs scramble ctrl at 3 min, kP<0.05 vs scramble ctrl at 15 min, n=5). (B) The viabilities of VECs were measured by MTT, and no significant changes were observed (n=3).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Control, Transfection

Journal: Acta pharmacologica Sinica

Article Title: Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn.

doi: 10.1038/aps.2011.142

Figure Lengend Snippet: Figure 6. Effects of integrin β4 knockdown in VECs on changes in ROCK levels in co-cultured VSMCs. VSMCs that had been co-cultured with VECs that were treated with or without integrin β4-specific siRNA (β4) or scramble control siRNA in the presence of SPC were immunostained for ROCK, α-SMA, or both. The bar graph shows the relative fluorescence intensity of ROCK per VSMC as determined by laser scanning confocal microscopy (bP<0.05 vs SPC-non-stimulated VSMCs; eP<0.05 vs SPC- stimulated scramble ctrl in co-cultured system, n=4).

Article Snippet: The primary antibodies (mouse anti-human integrin β4 or Fyn and rabbit anti-human ROCK), secondary antibodies (goat antimouse TR antibody and goat anti-rabbit FITC antibody) and the specific small interfering RNA (siRNA) oligonucleotides of human integrin β4 (SC-35678) were all purchased from the Santa Cruz Co, USA.

Techniques: Knockdown, Cell Culture, Control, Fluorescence, Confocal Microscopy

Journal: Nature Communications

Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

doi: 10.1038/ncomms10594

Figure Lengend Snippet: ( a ) Protein structure of a brain-enriched SNX protein, ARHGAP33. ARHGAP33 has an N-terminal PX domain, an SH3 domain and a RhoGAP domain. ( b , c ) Decreased cell-surface expression of TrkB in ARHGAP33 KO mice. Biotinylated cell-surface proteins (upper) and total lysates (lower) of WT and ARHGAP33 KO neurons (14 DIV) were immunoblotted with anti-TrkB, anti-TrkC, anti-SORT1, anti-GAPDH and anti-ARHGAP33 antibodies. ( b ) Representative blots. ( c ) Quantification of surface expression (each, n =8; TrkB, P =7.8 × 10 −4 ; TrkC and SORT1, P >0.05; Mann–Whitney U -test). The expression levels of TrkB, TrkC and SORT1 in ARHGAP33 KO neurons were normalized to those in WT neurons (The averaged WT values were set to 100%). ( d , e ) Decreased TrkB in the isolated PSD fraction of ARHGAP33 KO mice. The isolated PSD fraction and total lysates of WT and ARHGAP33 KO mice were immunoblotted with anti-TrkB, anti-PSD-95, anti-SORT1, and anti-ARHGAP33 antibodies. Representative blots ( d ). Quantification for the isolated PSD fraction (each, n =8, TrkB, P =7.7 × 10 −4 ; SORT1 and PSD-95, P >0.05; Mann–Whitney U -test) and for the total lysate (each, n =8, P >0.05; Mann–Whitney U -test; e ) The expression levels of TrkB, SORT1 and PSD-95 in the PSD fraction and total lysate from ARHGAP33 KO mice were normalized to those from WT mice (The averaged WT values were set to 100%). Note that the amounts of PSD-95 and SORT1 in the isolated PSD fraction from ARHGAP33 KO mice were not significantly different from those in the fraction from WT mice. * P <0.05. NS, not significant. Bars show median values. All western blots show representative results from eight independent experiments performed using different mice.

Article Snippet: The TaqMan Pre-Developed Assay Reagent kit (Applied Biosystems) was used for the mRNA expression analysis of GAPDH (4326317E), ARHGAP33 (Hs00364775_m1) and SORT1 (Hs00361747_m1).

Techniques: Expressing, MANN-WHITNEY, Isolation, Western Blot

Journal: Nature Communications

Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

doi: 10.1038/ncomms10594

Figure Lengend Snippet: ( a ) ARHGAP33 was localized to the Golgi apparatus. Double immunostaining for ARHGAP33 and a Golgi marker, GM130, in dissociated hippocampal neurons. Scale bar, 5 μm. Asterisks indicate the nucleus of neurons. Note that ARHGAP33 immunoreactivity was not detected in the neurons from ARHGAP33 KO mice (lower). The data are representative of three independent experiments. ( b ) ARHGAP33 and SORT1 were co-fractionated with a Golgi marker, GM130. Biochemical preparation of the Golgi membrane fraction from a mouse brain lysate with a discontinuous sucrose density gradient. Equal amounts of protein were loaded into individual lanes and probed with antibodies against SORT1, ARHGAP33, GM130 (a Golgi marker) and EEA1 (an endosomal marker). ( c ) The amount of TrkB, but not SORT1, in the Golgi membrane-enriched fraction was significantly increased in ARHGAP33 KO mice. Equal amount of the Golgi membrane fractions from WT and ARHGAP33 KO mice were probed with anti-TrkB, anti-SORT1, anti-GM130 and anti-ARHGAP33 antibodies. ( d ) Quantification of the amount of TrkB and SORT1 in the Golgi membrane-enriched fraction (each, n =7, TrkB, P =0.0017; SORT1, P >0.05, Mann–Whitney U -test). The levels of TrkB and SORT1 in the Golgi-enriched fraction from ARHGAP33 KO mice were normalized to those from WT mice (The averaged WT values were set to 100%). * P <0.05. NS, not significant. Bars show median values.

Article Snippet: The TaqMan Pre-Developed Assay Reagent kit (Applied Biosystems) was used for the mRNA expression analysis of GAPDH (4326317E), ARHGAP33 (Hs00364775_m1) and SORT1 (Hs00361747_m1).

Techniques: Double Immunostaining, Marker, Membrane, MANN-WHITNEY

Journal: Nature Communications

Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

doi: 10.1038/ncomms10594

Figure Lengend Snippet: ( a ) ARHGAP33 formed complexes with SORT1 and TrkB. ARHGAP33-immunoprecipitates (left) and hippocampal total lysates (right) were immunoblotted with the indicated antibodies. ( b – d ) ARHGAP33 formed complexes with SORT1 and TrkB. HEK293T cells were transfected with ARHGAP33, SORT1 and TrkB, as indicated. Immunoprecipitates were immunoblotted with the indicated antibodies. ( e ) Weakened interaction between TrkB and SORT1 in ARHGAP33 KO mice. SORT1 immunoprecipitates were immunoblotted with the indicated antibodies. Representative blots (left) and quantification of co-immunoprecipitated TrkB (right; each n =9; P =3.4 × 10 −4 , Mann–Whitney U -test). * P <0.05. The level of co-precipitated TrkB in ARHGAP33 KO mice was normalized to that in WT mice (the averaged WT value was set to 100%). Bars show median values. Western blots show representative results from nine independent experiments performed using different mice. ( f ) Weakened interaction between ARHGAP33 and TrkB in the SORT1 knockdown neuron. ARHGAP33-immunoprecipitates and total lysates were immunoblotted with the indicated antibodies. Representative blots (left), quantification of co-immunoprecipitated TrkB (centre) and quantification of SORT1 expression (confirmation of SORT1 knockdown; right; each n =8; TrkB, P =7.5 × 10 −4 , SORT1, P =7.7 × 10 −4 , Mann–Whitney U -test). The averaged values of the control neurons were set to 100%. * P <0.05. Bars show median values. Western blots show representative results from eight independent experiments performed using neurons from different mice; cont., control; KD, knockdown. Note that the MISSION shRNA construct (TRCN0000034496) was used. ( g ) In the ARHGAP33/SORT1/TrkB complex, ARHGAP33 promoted the interaction between TrkB and SORT1. In addition, ARHGAP33 is suggested to recruit Cdc42 into the complex . ( h ) Colocalization of ARHGAP33, SORT1 and TrkB at the perinuclear region. Triple immunostaining for ARHGAP33, SORT1 and TrkB in dissociated hippocampal neurons. Scale bar, 5 μm. The asterisks indicate the nucleus of the neuron. The arrowhead indicates the colocalization of these three proteins at the perinuclear region. The data are representative of five independent experiments. ( i ) Interactions between ARHGAP33 and Rab11 or Rab27 were not detected in the hippocampal lysates. ARHGAP33-immunoprecipitates (right) and hippocampal total lysates (left) were immunoblotted with the indicated antibodies.

Article Snippet: The TaqMan Pre-Developed Assay Reagent kit (Applied Biosystems) was used for the mRNA expression analysis of GAPDH (4326317E), ARHGAP33 (Hs00364775_m1) and SORT1 (Hs00361747_m1).

Techniques: Transfection, Immunoprecipitation, MANN-WHITNEY, Western Blot, Knockdown, Expressing, Control, shRNA, Construct, Triple Immunostaining

Journal: Nature Communications

Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

doi: 10.1038/ncomms10594

Figure Lengend Snippet: ( a , b ) Increased TrkB surface expression by ARHGAP33 and SORT1. HEK293T cells were transfected with TrkB, ARHGAP33 and SORT1 as indicated in b (1–4). Representative data from eight independent experiments of immunostaining of surface TrkB in HEK293T cells with an antibody against the extracellular region of TrkB ( a ). Scale bar, 10 μm. Representative blots (left) and the quantification of the surface TrkB (right) ( b ). Biotinylated cell-surface proteins were immunoblotted with the indicated antibodies. The surface expression of TrkB was enhanced by simultaneous expression of ARHGAP33 and SORT1 compared with the expression of ARHGAP33 alone (each n =8; ARHGAP33 alone versus ARHGAP33 and SORT1, P =0.004, Kruskal–Wallis test followed by post hoc Steel–Dwass tests). Western blots show representative results from eight independent experiments. The averaged value of surface TrkB level in cells expressing ARHGAP33 alone (lane 3) was set to 100%. * P <0.05. Bars show median values. ( c ) Requirement of SORT1 in ARHGAP33-mediated TrkB trafficking. Biotinylated cell-surface proteins were immunoblotted with the indicated antibodies. Representative blots (left), quantification of surface TrkB expression (centre) and quantification of SORT1 expression (confirmation of SORT1 knockdown; right; each n =10; surface TrkB, WT versus ARHGAP33 KO in control neurons, corrected P =6.0 × 10 −4 ; WT versus ARHGAP33 KO in SORT1 knockdown neurons, corrected P >0.05; ARHGAP33 KO versus ARHGAP33 KO plus SORT1 knockdown, corrected P >0.05, Mann–Whitney U -test with the Ryan's correction). Western blots show representative results from 10 independent experiments performed using neurons from different mice. The averaged values of WT mice in the control neurons were set to 100%. * P <0.05; cont., control; KD, knockdown; NS, not significant. Bars show median values. Note that the MISSION shRNA construct (TRCN0000034496) was used. ( d ) Strongly correlated expression of SORT1 and ARHGAP33 in immortalized lymphocytes from human blood (r=0.42, P <0.001, Spearman's rank order correlation test). Quantitative RT-PCR analysis of ARHGAP33 and SORT1 expression in immortalized lymphocytes was performed. Then, the levels of SORT1 and ARHGAP33 expression in each sample were plotted.

Article Snippet: The TaqMan Pre-Developed Assay Reagent kit (Applied Biosystems) was used for the mRNA expression analysis of GAPDH (4326317E), ARHGAP33 (Hs00364775_m1) and SORT1 (Hs00361747_m1).

Techniques: Expressing, Transfection, Immunostaining, Western Blot, Knockdown, Control, MANN-WHITNEY, shRNA, Construct, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

doi: 10.1038/ncomms10594

Figure Lengend Snippet: ( a , b ) Quantitative RT–PCR analysis of ARHGAP33 and SORT1 expression in immortalized lymphocytes from schizophrenia patients (schizo.) and age- and sex-matched controls. Significant reductions are observed in ARHGAP33 (each n =45, U =699, P =0.011, Mann–Whitney U -test; a ) and SORT1 (each n =45; U =683, P =7.8 × 10 −3 , Mann–Whitney U -test; b ) in schizophrenia patients. The expression levels of these genes were normalized to GAPDH mRNA. Bars show median values. ( c ) Linkage disequilibrium of ARHGAP33 in the HapMap JPT. Each diamond represents the correlation ( r 2 ) between each pair of SNPs, with darker shades representing stronger linkage disequilibrium, as obtained from the HapMap JST samples. ( d ) The locations of the SNPs analysed in this study. ( e , f ) Impact of the risk-T-allele on grey matter volume of the left middle temporal gyrus in schizophrenia patients. A significant cluster of the genotype effect in the left middle temporal gyrus is observed in schizophrenia patients, which is shown as cross-hairline (uncorrected P <0.001, cluster size >100; e ). Relative grey matter volumes extracted from the left middle temporal gyrus (F 1,122 =13.5, P =3.6 × 10 −4 , ANOVA), the right medial frontal gyrus (F 1,122 =13.5, P =8.2 × 10 −4 , ANOVA) and the right inferior temporal gyrus (F 1,122 =13.1, P =4.4 × 10 −4 , ANOVA; f ). * P <0.05. Data are expressed as the mean±s.e.m.

Article Snippet: The TaqMan Pre-Developed Assay Reagent kit (Applied Biosystems) was used for the mRNA expression analysis of GAPDH (4326317E), ARHGAP33 (Hs00364775_m1) and SORT1 (Hs00361747_m1).

Techniques: Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Kae prevents the invasion of SARS-CoV-2 particles into host cells. (A and B), immunofluorescence staining (A) or enzyme-linked immunosorbent assay (ELISA) analysis (B) of SARS-CoV-2 virus-like particles (VLPs) invasion into human induced pluripotent stem cells (hiPSC)-derived alveolar epithelial cells type II (AECII). Control: dimethyl sulfoxide (DMSO)-treated cells; spike (S) protein neutralizing antibody (S NAb): positive control; and Kae: 10 µM in DMSO.(C and D), the entry of SARS-CoV-2 VLPs into lungs of hACE2 transgenic mice evaluated by immunofluorescence staining (C) or luciferase activity (D).Control: 0.5% carboxymethylcellulose sodium-treated mice; ursodeoxycholic acid (UDCA): positive control; and Kae: indicated dosages in 0.5% carboxymethylcellulose sodium. (A and C), VLPs, ACE2, F-actin and nucleus are shown as indicated colors. Scale bar = 25 µm. (B), ***, p < 0.005 vs Blank. #, p < 0.05 and ##, p < 0.01 vs Control. (D), n = 4, ***, p < 0.005 vs Blank. ##, p < 0.01; and ###, p < 0.005 vs Control. ns: no statistical differences vs Control.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Virus, Derivative Assay, Control, Positive Control, Transgenic Assay, Luciferase, Activity Assay

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Kae targets coronaviral fusion. (A), flow cytometry analyses of the binding affinity between spike (S) protein receptor-binding domain (S-RBD) and HEK-293F cells expressing angiotensin-converting enzyme 2 (ACE2) in the presence or absence of Kae with the S protein neutralizing antibody (S NAb) as a positive control. ***, p < 0.005 vs Blank. ###, p < 0.005, and ns, no statistical differences vs Control. (B), impacts of Kae on SARS-CoV-2 endocytosis into Vero E6 cells with S NAb as a positive control. ***, p < 0.005, and ns, no statistical differences vs Control. (C, D and E), effects of Kae on membrane fusion mediated by SARS-CoV-2 variants (C), or SARS-CoV (D), or MERS-CoV (E) with transmembrane serine protease 2 (TMPRSS2) inhibitor, camostat mesylate (CM), as a positive control. Scale bar = 300 µm. **, p < 0.01; and ***, p < 0.005 vs Control. Control: dimethyl sulfoxide (DMSO)-treated cells; DPP4: dipeptidyl peptidase 4; ns: no statistical differences vs Control.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Flow Cytometry, Binding Assay, Expressing, Positive Control, Control, Membrane

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Kae interferes with the S2 subunit of SARS-CoV-2 spike protein. (A), immunoblots representing the cleavage and abundance of SARS-CoV-2 spike (S) protein induced by Kae, with camostat mesylate (CM) as a positive control. (B), thermal stability determinations for full-length S protein, S1 and S2 subunits of SARS-CoV-2 in the presence or absence of Kae. Control: dimethyl sulfoxide (DMSO)-treated cells.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Western Blot, Positive Control, Control

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Kae impairs HR1 and HR2 of SARS-CoV-2 S2 subunit. (A and B), circular dichroism (CD) spectra analyses of synthesized heptad repeat 1 (HR1) and heptad repeat 2 (HR2) peptide, the formation of six-helix bundle (6-HB, HR1+HR2), and impacts of Kae on 6-HB, HR1 and HR2 using isopropanol as solvent controls. (C, D, E and F), native polyacrylamide gel electrophoresis (N-PAGE) assessment of effects of Kae on HR1, HR2 and 6-HB. *, p < 0.05; **, p < 0.01; and ***, p < 0.005 vs Control. ns: no statistical differences vs Blank. (E and F), the stability of wildtype (WT) and mutant (MT) HR2 in the absence (E) or presence (F) of Kae compared to corresponding wildtype peptide. (G), atomic force microscopy (AFM) topographic images before and after adding Kae to HR1, HR2 and 6-HB. Scale bar = 4 µm. (C, D, F and G), Control: dimethyl sulfoxide (DMSO)-treated peptides.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Circular Dichroism, Synthesized, Solvent, Polyacrylamide Gel Electrophoresis, Control, Mutagenesis, Microscopy

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Schematic diagram of the inhibitory mechanism of Kae against SARS-CoV-2 invasion. Instead of blocking the interaction between angiotensin-converting enzyme 2 (ACE2) and spike (S) protein receptor-binding domain (S-RBD), Kae disrupts heptad repeat 1 (HR1) and heptad repeat 2 (HR2) segments in the S2 subunit of SARS-CoV-2 S protein, leading to inhibitions of viral fusion-mediated invasion of SARS-CoV-2.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Blocking Assay, Binding Assay

Journal: Scientific Reports

Article Title: HNF4A ameliorates acute liver failure by inhibiting NCOA4-mediated ferritinophagy

doi: 10.1038/s41598-025-13798-3

Figure Lengend Snippet: Impact of siNCOA4 on cellular responses in LO2 cells. A: NCOA4 mRNA expression in LO2 cells after siNCOA4 transfection (detected by RT-PCR); silencing efficiency was verified ( P < 0.01). B: NCOA4 protein expression in LO2 cells after siNCOA4 transfection (detected by Western Blot); silencing efficiency was confirmed ( P = 0.03). C: Cell viability in control, siNCOA4, LPS, and LPS + siNCOA4 groups (assessed by CCK-8 assay); siNCOA4 reversed LPS-induced viability reduction ( P = 0.020, one-way ANOVA). D: Mitochondrial morphology in each group (observed by transmission electron microscopy, scale bar: 10 μm); siNCOA4 alleviated LPS-induced mitochondrial deformation. E: Intracellular Fe²⁺ levels (detected by FerroOrange staining, scale bar: 100 μm); siNCOA4 reduced LPS-induced Fe²⁺ accumulation ( P = 0.015, one-way ANOVA). F: Mitochondrial membrane potential (assessed by JC-1 fluorescence, scale bar: 100 μm); siNCOA4 restored LPS-reduced membrane potential ( P = 0.018, one-way ANOVA). n = 5.

Article Snippet: For NCOA4 silencing, LO2 cells were seeded in 6-well plates at 5 × 105 cells/well and cultured until 60–70% confluence. siNCOA4 (NCOA4 Human siRNA Oligo Duplex, SR322322, Origene) was transfected using LipofectamineTM RNAiMAX Transfection Reagent (13778030, Invitrogen) at a final concentration of 50 nM, following the manufacturer’s protocol (siRNA: transfection reagent = 1:2, v/w).

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, CCK-8 Assay, Transmission Assay, Electron Microscopy, Staining, Membrane, Fluorescence

Journal: Scientific Reports

Article Title: HNF4A ameliorates acute liver failure by inhibiting NCOA4-mediated ferritinophagy

doi: 10.1038/s41598-025-13798-3

Figure Lengend Snippet: Biochemical changes induced by siNCOA4 in different groups. A: Relative GSH levels in each group; siNCOA4 increased GSH in LPS-treated cells ( P = 0.022, one-way ANOVA). B: MDA levels ratio in each group; siNCOA4 decreased MDA in LPS-treated cells ( P = 0.019, one-way ANOVA). C: Intracellular ROS levels (detected by DCFH-DA and flow cytometry); siNCOA4 reversed LPS-induced ROS accumulation ( P < 0.01). D: GPX4 mRNA expression in each group (detected by RT-PCR); siNCOA4 upregulated GPX4 in LPS-treated cells ( P = 0.016, one-way ANOVA). E: SLC7A11 mRNA expression in each group (detected by RT-PCR); siNCOA4 upregulated SLC7A11 in LPS-treated cells ( P = 0.017, one-way ANOVA). F: GPX4 and SLC7A11 protein expression (detected by Western Blot); siNCOA4 increased both proteins in LPS-treated cells ( P < 0.02, one-way ANOVA). n = 5.

Article Snippet: For NCOA4 silencing, LO2 cells were seeded in 6-well plates at 5 × 105 cells/well and cultured until 60–70% confluence. siNCOA4 (NCOA4 Human siRNA Oligo Duplex, SR322322, Origene) was transfected using LipofectamineTM RNAiMAX Transfection Reagent (13778030, Invitrogen) at a final concentration of 50 nM, following the manufacturer’s protocol (siRNA: transfection reagent = 1:2, v/w).

Techniques: Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Scientific Reports

Article Title: HNF4A ameliorates acute liver failure by inhibiting NCOA4-mediated ferritinophagy

doi: 10.1038/s41598-025-13798-3

Figure Lengend Snippet: Impact of siNCOA4 and HNF4A Overexpression (oeHNF4A) on Cellular Responses. A: Cell viability in control, LPS, LPS + oeHNF4A, LPS + oeHNF4A + siNCOA4, and LPS + oeHNF4A + oeNCOA4 groups; combined oeHNF4A and siNCOA4 synergistically increased viability ( P = 0.010, one-way ANOVA). B: Intracellular Fe²⁺ levels (scale bar: 100 μm); combined treatment reduced LPS-induced Fe²⁺ deposition ( P = 0.012, one-way ANOVA). C: Mitochondrial membrane potential (JC-1 fluorescence); combined treatment restored LPS-reduced potential ( P = 0.014, one-way ANOVA). D: GSH levels; combined treatment increased GSH in LPS-treated cells ( P = 0.016, one-way ANOVA). E: MDA ratio; combined treatment decreased MDA in LPS-treated cells ( P = 0.018, one-way ANOVA). F: Intracellular ROS levels; combined treatment reduced LPS-induced ROS ( P = 0.015, one-way ANOVA). G: GPX4 mRNA expression; combined treatment upregulated GPX4 ( P = 0.013, one-way ANOVA). H: SLC7A11 mRNA expression; combined treatment upregulated SLC7A11 ( P = 0.014, one-way ANOVA). I: GPX4 and SLC7A11 protein expression; combined treatment increased both proteins ( P < 0.02, one-way ANOVA). n = 5.

Article Snippet: For NCOA4 silencing, LO2 cells were seeded in 6-well plates at 5 × 105 cells/well and cultured until 60–70% confluence. siNCOA4 (NCOA4 Human siRNA Oligo Duplex, SR322322, Origene) was transfected using LipofectamineTM RNAiMAX Transfection Reagent (13778030, Invitrogen) at a final concentration of 50 nM, following the manufacturer’s protocol (siRNA: transfection reagent = 1:2, v/w).

Techniques: Over Expression, Control, Membrane, Fluorescence, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Validation of Hv 1 channel functions in BV2 microglial cells using small molecule modulators

doi: 10.3389/fncel.2025.1624224

Figure Lengend Snippet: Expression of Hv 1 channels in BV2 microglial cells. (A) Representative immunocytochemistry images of scramble shRNA transduced cells (control) and Hv 1 shRNA transduced cells stained with anti-Hv 1 antibody (red) and DAPI (blue). Scale bar = 40 μM (B) A comparison of Hv 1 channel protein expression by western blot in control cells and in Hv 1 shRNA transduced cells. (C) Hv 1 mRNA expression in control and Hv 1 shRNA cells was detected by using RT-PCR. (D) Representative whole-cell currents recorded in response to 3 s long pulses, stepping from a holding potential of −60 mV to levels ranging from −80 to +100 mV, with 20 mV increments in control (black traces) and Hv 1 shRNA cells (blue traces). (E) Mean current density versus membrane potential curves corresponding to the control condition and Hv 1 shRNA condition is plotted. Current density was measured by normalizing maximal peak currents with cell capacitance (pA/pF). *** p < 0.001, two-tailed unpaired t-test, N = 3.

Article Snippet: 10 μl of Lentiviral shRNA particles (mouse HVCN1; Origene-TL504897V and scramble; Origene–TR30021V) were added in culture medium with polybrene (final concentration 8 μg/ml) to the total volume of 1 ml DMEM/F-12 media and left for 72 h. Finally, the transduction efficiency was evaluated under the fluorescent microscope and gene knockdown was validated using immunocytochemistry, qPCR, western blot and patch clamp electrophysiology techniques.

Techniques: Expressing, Immunocytochemistry, shRNA, Control, Staining, Comparison, Western Blot, Reverse Transcription Polymerase Chain Reaction, Membrane, Two Tailed Test

Journal: Cell Stress & Chaperones

Article Title: HSP70 inhibits stress-induced cardiomyocyte apoptosis by competitively binding to FAF1

doi: 10.1007/s12192-015-0589-9

Figure Lengend Snippet: FAF1 levels increased with stress intensity, and overexpression of FAF1 exacerbated stress-induced apoptosis. a After restraint stress, whole-cell lysates of rat myocardia were subjected to western blot analysis using an anti-FAF1 antibody. GAPDH was used as a loading control. b Levels of FAF1 in H9C2 cells treated with different concentrations of NE for 36 h. c After transfection with the pcDNA3.1(+)-rFAF1 plasmid, the activity of caspase-8 in whole-cell lysates was detected using a Caspase-8 Activity Assay Kit. d Flow cytometric analysis of apoptosis. The data shown represent the mean ± SD from three independent experiments. *P < 0.05 vs the control group. #P < 0.05 vs the NE group

Article Snippet: After incubation with 5 % BSA for 2 h at room temperature, the membrane was incubated at 4 °C overnight with one of the following primary antibodies: mouse monoclonal antibodies to HSP70 (Cell Signaling Technology, USA), FAS (Boster Biotechnology Company, China), or GAPDH (Sungene Biotech Company, China) or a goat polyclonal antibody to FAF1 (Santa Cruz Biotechnology, Inc., CA, USA).

Techniques: Over Expression, Western Blot, Control, Transfection, Plasmid Preparation, Activity Assay

Journal: Cell Stress & Chaperones

Article Title: HSP70 inhibits stress-induced cardiomyocyte apoptosis by competitively binding to FAF1

doi: 10.1007/s12192-015-0589-9

Figure Lengend Snippet: HSP70 competitively inhibited the binding of FAF1 to FAS in cardiac H9C2 cells. a H9C2 cells were lysed and immunoprecipitated (IP) with anti-hFAF1 polyclonal antibody and mouse IgG as a control. The precipitates were analyzed by SDS-PAGE and western blot analysis using anti-FAF1 polyclonal antibody (upper panel) and anti-HSP70 monoclonal antibody (lower panel). b FAF1-FAS binding was analyzed by western blot using an anti-FAF1 monoclonal antibody (upper panel) after immunoprecipitation with an anti-FAS polyclonal antibody (lower panel). c After the pEGFP-HSP70△N-(121–642) and pEGFP-HSP70-(1–642) plasmids were transfected into H9C2 cells, cell lysates were immunoprecipitated with anti-FAF1 polyclonal antibody (upper panel) and anti-GFP monoclonal antibody (lower panel). d The activity of caspase-8 was measured with a Caspase-8 Activity Assay Kit. e The apoptosis of H9C2 cells transfected with HSP70 and the HSP70 mutant was measured by staining with 7-AAD and Annexin V-PE followed by FCM analysis. The data shown represent the mean ± SD from three independent experiments. *P < 0.05 vs the control group. #P < 0.05 vs the NE group

Article Snippet: After incubation with 5 % BSA for 2 h at room temperature, the membrane was incubated at 4 °C overnight with one of the following primary antibodies: mouse monoclonal antibodies to HSP70 (Cell Signaling Technology, USA), FAS (Boster Biotechnology Company, China), or GAPDH (Sungene Biotech Company, China) or a goat polyclonal antibody to FAF1 (Santa Cruz Biotechnology, Inc., CA, USA).

Techniques: Binding Assay, Immunoprecipitation, Control, SDS Page, Western Blot, Transfection, Activity Assay, Mutagenesis, Staining

Journal: Frontiers in Immunology

Article Title: A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice

doi: 10.3389/fimmu.2023.1224634

Figure Lengend Snippet: Inactivated whole-virion (WV) vaccine retains the protein composition and the structure of the original virus. (A) Scheme of inactivated WV (Gamma strain) production. (B) VeroE6/TMPRSS2 cells were treated with medium as a negative control, inactivated WV (undiluted) and live virus (diluted 10 4 -fold with medium) at 37 °C for 3 days, followed by plaque counting. (C, D) Inactivated WV were validated using (C) SDS-PAGE; left: marker, right: sample and (D) western blotting; left: marker, right: S protein and M protein. Each experiment was performed in duplicate. (E) TEM micrograph of SARS-CoV-2 virus particles, Scale bar, 100 nm.

Article Snippet: The transferred membrane was incubated with rabbit anti-SARS-CoV-2 (2019-nCoV) spike antibody (Polyclonal IgG; Sino Biological).

Techniques: Virus, Negative Control, SDS Page, Marker, Western Blot

Journal: Frontiers in Immunology

Article Title: A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice

doi: 10.3389/fimmu.2023.1224634

Figure Lengend Snippet: Nasal administration of the inactivated whole-virion (WV) vaccine induces S protein-specific IgG in serum and IgA in nasal wash. (A–H) Mice were immunized twice with inactivated WV intranasally (i.n.) or with inactivated WV and alum subcutaneously (s.c.). Unimmunized mice were used as controls (cont.). (A–F) Endpoint titers of S protein-specific IgG in (A) serum, (B) bronchoalveolar lavage fluid (BALF), and (C) nasal wash, and endpoint titers of S protein-specific IgA in (D) serum, (E) BALF, and (F) nasal wash were evaluated. (G, H) Measurement of neutralization against vesicular stomatitis virus-based pseudotyped viruses displaying Gamma spike of SARS-CoV-2 in (G) serum samples and (H) nasal wash samples. The significance of differences in the (G) 50-fold-diluted serum samples and (H) 4-fold-diluted nasal wash samples was evaluated. The data is presented as mean ± SD. Each experiment was performed in duplicate. (A–H) n = 5 per group. (A–H) * P < 0.05; ** P < 0.01 as indicated by Tukey’s test. ns, not statistically significant.

Article Snippet: The transferred membrane was incubated with rabbit anti-SARS-CoV-2 (2019-nCoV) spike antibody (Polyclonal IgG; Sino Biological).

Techniques: Neutralization, Virus

Journal: Frontiers in Immunology

Article Title: A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice

doi: 10.3389/fimmu.2023.1224634

Figure Lengend Snippet: Nasal administration of the inactivated whole-virion (WV) vaccine protects against upper and lower respiratory tract infections caused by SARS-CoV-2. (A–E) Mice were immunized twice with inactivated whole-virion (WV) vaccine intranasally (i.n.) or with inactivated WV and alum subcutaneously (s.c.). Unimmunized mice were used as controls (cont.). (A) Mice were intranasally infected with SARS-CoV-2 MA10 (5 × 10 4 PFU) in 5 μL of PBS and the SARS-CoV-2 titer in nasal turbinate was assayed using the plaque assay. (B–E) Mice were intranasally infected with SARS-CoV-2 MA10 ( B, C ; 5 × 10 4 PFU/mouse, D, E ; 2 × 10 5 PFU/mouse) in 20 μL of PBS; (B, D) body weight loss and (C, E) survival were monitored. The data is presented as the mean ± SD. Each experiment was performed in duplicate. (A) n = 5 per group. (B, C) n = 4–5 per group. (D, E) n = 10 per group. (A, B, D) * P < 0.05; ** P < 0.01 as indicated by Tukey’s test. (C, E) ** P < 0.01 vs. the control group as indicated by comparing Kaplan–Meier curves using the Log rank test.

Article Snippet: The transferred membrane was incubated with rabbit anti-SARS-CoV-2 (2019-nCoV) spike antibody (Polyclonal IgG; Sino Biological).

Techniques: Infection, Plaque Assay

Journal: Frontiers in Immunology

Article Title: A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice

doi: 10.3389/fimmu.2023.1224634

Figure Lengend Snippet: Systemic priming by mRNA vaccine followed by intranasal boosting with inactivated whole-virion (WV) vaccine induces S protein-specific IgG in serum and IgA in nasal wash. (A–C) Mice were immunized intranasally (i.n.) with inactivated WV or intramuscularly (i.m.) with 1 μg of mRNA vaccine encoding SARS-CoV-2 Spike. (A) Graphical illustration of the experimental procedure. (B, C) Endpoint titers of S protein-specific (B) IgA in the nasal wash and (C) IgG in the serum were evaluated. The data is presented as the mean ± SD. Each experiment was performed in duplicate. (B, C) n = 7–11 per group. (B, C) ** P < 0.01 as indicated by Tukey’s test. ns, not statistically significant.

Article Snippet: The transferred membrane was incubated with rabbit anti-SARS-CoV-2 (2019-nCoV) spike antibody (Polyclonal IgG; Sino Biological).

Techniques:

Journal: Brain Pathology

Article Title: Clusterin contributes to early stage of Alzheimer's disease pathogenesis

doi: 10.1111/bpa.12660

Figure Lengend Snippet: Effect of clusterin and age on expression of neuronal or synaptic proteins in the brain of 5×FAD mouse. A. Representative Western blots show expression of neuronal or synaptic proteins in 5‐ and 10‐month‐old clu +/+;5×FAD and clu −/−;5×FAD mice and 5‐month‐old nontransgenic wild‐type (WT) mice, representing only results with significant differences in expression; neuronal specific NeuN (molecular size, 46‐48 kD), nonphosphorylated neurofilament SMI‐32 (200 kD), presynaptic synaptophysin (38 kD), axon initial segment‐specific and voltage‐gated sodium channels‐associated ankyrin G (190 kD), postsynaptic PSD‐95 (95 kD), vesicle‐associated membrane protein (VAMP, synaptobrevin; 16 kD), postsynaptic scaffold protein ProSAP2/Shank3 (180 kD) and ß‐actin (42 kD). B–H. Densitometric quantification of indicated proteins after normalization to ß‐actin. Graphs show significant differences in the levels of the indicated proteins (arrows in blots) between nontransgenic wild‐type and 5×FAD mice and between 5‐month‐old clu +/+;5×FAD and clu −/−;5×FAD mice but not between 10‐month‐old clu +/+;5×FAD and clu −/−;5×FAD mice. Protein levels are shown as values relative to a control (black bars, wild‐type mice). Bars denote mean ± SEM of three independent experiments. Each group includes 6 mice (3 males and 3 females). *P < 0.05, **P < 0.01, or ***P < 0.001.

Article Snippet: The following primary antibodies were used for immunoblotting: anti‐Aß(1–16) (6E10; BioLegend; dilution, 1:2000), anti‐clusterin/Apo J (Novus Biologicals; 1:1000), anti‐apolipoprotein E (Apo E; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1000), anti‐DKK‐1 (Dickkopf‐1; Boster, Pleasanton, CA, USA: 1:1000), anti‐NeuN (Merck Millipore, Darmstadt, Germany; 1:500), anti‐nonphosphorylated neurofilament protein SMI‐32 (BioLegend; 1:2000), anti‐synaptophysin (Santa Cruz Biotechnology; 1:5000), anti‐ankyrin G (Santa Cruz Biotechnology; 1:2000), anti‐PSD95 (postsynaptic density protein 95; Applied Biological Materials, Richmond, Canada; 1:2000), anti‐VAMP (vesicle‐associated membrane protein)/synaptobrevin (Santa Cruz Biotechnology; 1:1000), anti‐ProSAP2 (postsynaptic scaffold protein)/Shank 3 (Santa Cruz Biotechnology; 1:500) and anti‐ß‐actin (Sigma‐Aldrich; 1:2000) antibodies.

Techniques: Expressing, Western Blot

Journal: Molecular Biology of the Cell

Article Title: SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria

doi: 10.1091/mbc.E13-01-0016

Figure Lengend Snippet: SARM1 binds to and recruits TRAF2/6 into PINK1 complexes. (A, B) Cell lysates from HEK293 cells cotransfected with vectors expressing TRAF6/2-Myc and SARM1-Flag, respectively, were immunoprecipitated with anti-Flag antibody and analyzed by Western blotting. (C) Cell lysates from HEK293 cells cotransfected with vectors expressing PINK1-HA and/or SARM1-Flag were immunoprecipitated with anti-Flag antibody and analyzed by Western blotting. Endogenous TRAF2 and TRAF6 were recovered in the immune complex of PINK1–SARM1.

Article Snippet: The antibodies used were as follows: rabbit polyclonal antibody against PINK1 (BC100-494; Novus Biologicals, Littleton, CO); rabbit polyclonal antibody against PINK1 raised in our previous study ( Murata et al. , 2011a ); antibodies against TRAF6 (rabbit monoclonal, 8028), LC-3B (rabbit polyclonal, 2775), Flag (rabbit polyclonal, 2368), Myc (mouse monoclonal, 2276), and HRP-labeled anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technologies, Danvers, MA); antibodies against TRAF2 (rabbit polyclonal, 592), Flag (rabbit polyclonal, PM020 for immunocytochemistry), and histidine (rabbit polyclonal, PM032) (MBL, Woburn, MA); rabbit polyclonal antibody against SARM1 (NB100-94383; Novus Biologicals); antibodies against ubiquitin (mouse monoclonal, sc-8017), Tom20 (mouse monoclonal, sc-17764), Tom20 (rabbit polyclonal, sc-11415), and Bcl-2 (mouse monoclonal, sc-7382; Santa Cruz Biotechnology, Santa Cruz, CA); antibodies against ubiquitin (mouse monoclonal, 550944) and cytochrome c (mouse monoclonal, 556433; BD PharMingen, San Jose, CA); antibodies against tubulin (mouse monoclonal, T5168) and HA-agarose (mouse monoclonal, A2095 for immunoprecipitation; Sigma-Aldrich); and HRP-labeled TrueBlot anti-mouse IgG (18-8817-33; eBioscience, San Diego, CA).

Techniques: Expressing, Immunoprecipitation, Western Blot

Journal: Molecular Biology of the Cell

Article Title: SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria

doi: 10.1091/mbc.E13-01-0016

Figure Lengend Snippet: SARM1 promotes TRAF6-mediated ubiquitination of PINK1. (A) Ubiquitination of PINK1 by TRAF6 in cells. Cell lysates from SH-SY5Y cells transfected with PINK1-HA and Flag-ubiquitin (Ub) along with TRAF2-Myc or TRAF6-Myc were immunoprecipitated with anti-HA agarose and analyzed by Western blotting. (B) An in vitro ubiquitination assay was performed by incubation of purified GST-PINK1 with Flag-tagged SARM1, Myc-tagged TRAF2, or Myc-tagged TRAF6 in the presence of E1, E2 (UbcH13/Uev1a), and ubiquitin. Ubiquitination of PINK1 was detected by pull down with glutathione-agarose beads, followed by Western blot analysis for ubiquitin. (C) SARM1 promotes TRAF6-mediated PINK1 ubiquitination in SH-SY5Y cells. SH-SY5Y cells were transfected with designated constructs. Levels of PINK1 ubiquitination were determined by immunoprecipitation with anti-HA agarose, followed by Western blot analysis. (D) Ubiquitination levels are reduced in PINK1 mutants. The experiment was performed under conditions similar to those in C.

Article Snippet: The antibodies used were as follows: rabbit polyclonal antibody against PINK1 (BC100-494; Novus Biologicals, Littleton, CO); rabbit polyclonal antibody against PINK1 raised in our previous study ( Murata et al. , 2011a ); antibodies against TRAF6 (rabbit monoclonal, 8028), LC-3B (rabbit polyclonal, 2775), Flag (rabbit polyclonal, 2368), Myc (mouse monoclonal, 2276), and HRP-labeled anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technologies, Danvers, MA); antibodies against TRAF2 (rabbit polyclonal, 592), Flag (rabbit polyclonal, PM020 for immunocytochemistry), and histidine (rabbit polyclonal, PM032) (MBL, Woburn, MA); rabbit polyclonal antibody against SARM1 (NB100-94383; Novus Biologicals); antibodies against ubiquitin (mouse monoclonal, sc-8017), Tom20 (mouse monoclonal, sc-17764), Tom20 (rabbit polyclonal, sc-11415), and Bcl-2 (mouse monoclonal, sc-7382; Santa Cruz Biotechnology, Santa Cruz, CA); antibodies against ubiquitin (mouse monoclonal, 550944) and cytochrome c (mouse monoclonal, 556433; BD PharMingen, San Jose, CA); antibodies against tubulin (mouse monoclonal, T5168) and HA-agarose (mouse monoclonal, A2095 for immunoprecipitation; Sigma-Aldrich); and HRP-labeled TrueBlot anti-mouse IgG (18-8817-33; eBioscience, San Diego, CA).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, In Vitro, Incubation, Purification, Construct

Journal: Molecular Biology of the Cell

Article Title: SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria

doi: 10.1091/mbc.E13-01-0016

Figure Lengend Snippet: PINK1 is ubiquitinated by TRAF6 upon impairment of mitochondria. (A) PINK1 is ubiquitinated under the condition of low membrane potential of mitochondria. SH-SY5Y cells were transfected with designated constructs for 48 h, followed by treatment with 10 μM CCCP for 6 h. Ubiquitination of PINK1 was determined by immunoprecipitation with anti-HA agarose, followed by Western blot analysis. (B) SH-SY5Y cells were treated with 10 μM CCCP for 6 h. Ubiquitination of endogenous PINK1 was determined by immunoprecipitation with anti-PINK1 antibody, followed by Western blot analysis. K63-linked ubiquitination of PINK1 was detected using biotinylated K63-TUBE1 and streptavidin-HRP. Arrowheads indicate immunoprecipitated PINK1. (C, D) Overexpression of dominant-negative TRAF6 destabilizes exogenous and endogenous PINK1 as shown in C and D, respectively. The experiment was performed under conditions similar to those in A. (E) Knockdown of TRAF6 inhibits stabilization of endogenous PINK1. SH-SY5Y cells were transfected with designated siRNAs for 72 h, followed by treatment with 10 μM CCCP for 6 h.

Article Snippet: The antibodies used were as follows: rabbit polyclonal antibody against PINK1 (BC100-494; Novus Biologicals, Littleton, CO); rabbit polyclonal antibody against PINK1 raised in our previous study ( Murata et al. , 2011a ); antibodies against TRAF6 (rabbit monoclonal, 8028), LC-3B (rabbit polyclonal, 2775), Flag (rabbit polyclonal, 2368), Myc (mouse monoclonal, 2276), and HRP-labeled anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technologies, Danvers, MA); antibodies against TRAF2 (rabbit polyclonal, 592), Flag (rabbit polyclonal, PM020 for immunocytochemistry), and histidine (rabbit polyclonal, PM032) (MBL, Woburn, MA); rabbit polyclonal antibody against SARM1 (NB100-94383; Novus Biologicals); antibodies against ubiquitin (mouse monoclonal, sc-8017), Tom20 (mouse monoclonal, sc-17764), Tom20 (rabbit polyclonal, sc-11415), and Bcl-2 (mouse monoclonal, sc-7382; Santa Cruz Biotechnology, Santa Cruz, CA); antibodies against ubiquitin (mouse monoclonal, 550944) and cytochrome c (mouse monoclonal, 556433; BD PharMingen, San Jose, CA); antibodies against tubulin (mouse monoclonal, T5168) and HA-agarose (mouse monoclonal, A2095 for immunoprecipitation; Sigma-Aldrich); and HRP-labeled TrueBlot anti-mouse IgG (18-8817-33; eBioscience, San Diego, CA).

Techniques: Membrane, Transfection, Construct, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Over Expression, Dominant Negative Mutation, Knockdown

Journal: Molecular Biology of the Cell

Article Title: SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria

doi: 10.1091/mbc.E13-01-0016

Figure Lengend Snippet: TRAF6 ubiquitinates PINK1 at Lys433. SH-SY5Y cells were transfected with designated constructs along with C-terminal HA-tagged PINK1 wild type or mutants with different lysine-to-arginine mutations for 48 h. (A) Ubiquitination of PINK1 was determined by immunoprecipitation with anti-HA agarose, followed by Western blot analysis. (B) The experiment was performed under conditions similar to those in A. For K433R, double amount of plasmid was used compared with that of PINK1-WT to attain the same protein levels of PINK1. (C, D) SH-SY5Y cells transfected with designated constructs were treated with 10 μM CCCP for 6 h (C) or 0.5 μM MG-132 for 12 h (D).

Article Snippet: The antibodies used were as follows: rabbit polyclonal antibody against PINK1 (BC100-494; Novus Biologicals, Littleton, CO); rabbit polyclonal antibody against PINK1 raised in our previous study ( Murata et al. , 2011a ); antibodies against TRAF6 (rabbit monoclonal, 8028), LC-3B (rabbit polyclonal, 2775), Flag (rabbit polyclonal, 2368), Myc (mouse monoclonal, 2276), and HRP-labeled anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technologies, Danvers, MA); antibodies against TRAF2 (rabbit polyclonal, 592), Flag (rabbit polyclonal, PM020 for immunocytochemistry), and histidine (rabbit polyclonal, PM032) (MBL, Woburn, MA); rabbit polyclonal antibody against SARM1 (NB100-94383; Novus Biologicals); antibodies against ubiquitin (mouse monoclonal, sc-8017), Tom20 (mouse monoclonal, sc-17764), Tom20 (rabbit polyclonal, sc-11415), and Bcl-2 (mouse monoclonal, sc-7382; Santa Cruz Biotechnology, Santa Cruz, CA); antibodies against ubiquitin (mouse monoclonal, 550944) and cytochrome c (mouse monoclonal, 556433; BD PharMingen, San Jose, CA); antibodies against tubulin (mouse monoclonal, T5168) and HA-agarose (mouse monoclonal, A2095 for immunoprecipitation; Sigma-Aldrich); and HRP-labeled TrueBlot anti-mouse IgG (18-8817-33; eBioscience, San Diego, CA).

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

Journal: Molecular Biology of the Cell

Article Title: SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria

doi: 10.1091/mbc.E13-01-0016

Figure Lengend Snippet: SARM1 and TRAF6 promote the translocation of PINK1 to the outer membrane of mitochondria. (A) SH-SY5Y cells were transfected with SARM1-His for 48 h, followed by treatment with 10 μM CCCP for 2 h. The cell lysates were immunoprecipitated with anti-Tom20 antibody or control mouse IgG, followed by Western blot analysis. (B) SARM1 and TRAF6 promote the association of PINK1 with Tom20. SH-SY5Y cells were transfected with designated constructs for 48 h and analyzed under the conditions similar to those in A. Arrowhead indicates immunoprecipitated PINK1. (C) PINK1-WT but not OPA3-PINK1 interacts with SARM1. Cell lysates from SH-SY5Y cells transfected with SARM1-His along with PINK1-WT-HA or OPA3-PINK1-HA (PINK1 1-110 fused to the N-terminal mitochondrial anchor of OPA3 at amino acid positions 1–30) were immunoprecipitated with anti-HA agarose and analyzed by Western blotting.

Article Snippet: The antibodies used were as follows: rabbit polyclonal antibody against PINK1 (BC100-494; Novus Biologicals, Littleton, CO); rabbit polyclonal antibody against PINK1 raised in our previous study ( Murata et al. , 2011a ); antibodies against TRAF6 (rabbit monoclonal, 8028), LC-3B (rabbit polyclonal, 2775), Flag (rabbit polyclonal, 2368), Myc (mouse monoclonal, 2276), and HRP-labeled anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technologies, Danvers, MA); antibodies against TRAF2 (rabbit polyclonal, 592), Flag (rabbit polyclonal, PM020 for immunocytochemistry), and histidine (rabbit polyclonal, PM032) (MBL, Woburn, MA); rabbit polyclonal antibody against SARM1 (NB100-94383; Novus Biologicals); antibodies against ubiquitin (mouse monoclonal, sc-8017), Tom20 (mouse monoclonal, sc-17764), Tom20 (rabbit polyclonal, sc-11415), and Bcl-2 (mouse monoclonal, sc-7382; Santa Cruz Biotechnology, Santa Cruz, CA); antibodies against ubiquitin (mouse monoclonal, 550944) and cytochrome c (mouse monoclonal, 556433; BD PharMingen, San Jose, CA); antibodies against tubulin (mouse monoclonal, T5168) and HA-agarose (mouse monoclonal, A2095 for immunoprecipitation; Sigma-Aldrich); and HRP-labeled TrueBlot anti-mouse IgG (18-8817-33; eBioscience, San Diego, CA).

Techniques: Translocation Assay, Membrane, Transfection, Immunoprecipitation, Control, Western Blot, Construct

Journal: Molecular Biology of the Cell

Article Title: SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria

doi: 10.1091/mbc.E13-01-0016

Figure Lengend Snippet: SARM1 and TRAF6 are required for recruitment of parkin to damaged mitochondria. SH-SY5Y (A) and HeLa cells (B) were transfected with Flag-parkin for 48 h, followed by treatment with 10 μM CCCP for 6 h and then immunostained with the indicated antibodies. (C–F) HeLa cells were transfected with designated constructs for 48 h. The number of cells with parkin-positive mitochondria among the parkin-expressing cells. Three independent experiments were performed, and at least 100 cells were counted for each experiment. Error bars represent mean ± SD. *Significantly different from the control group ( p < 0.01); ns, not significant. (C) Cells were transfected with PINK1 variants. (D) Effect of cotransfection with SARM1 and TRAF6. (E) Effect of knockdown of endogenous PINK1, SARM1, TRAF2, and TRAF6. (F) A rescue experiment for down-regulated endogenous proteins with overexpression of the respective proteins.

Article Snippet: The antibodies used were as follows: rabbit polyclonal antibody against PINK1 (BC100-494; Novus Biologicals, Littleton, CO); rabbit polyclonal antibody against PINK1 raised in our previous study ( Murata et al. , 2011a ); antibodies against TRAF6 (rabbit monoclonal, 8028), LC-3B (rabbit polyclonal, 2775), Flag (rabbit polyclonal, 2368), Myc (mouse monoclonal, 2276), and HRP-labeled anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technologies, Danvers, MA); antibodies against TRAF2 (rabbit polyclonal, 592), Flag (rabbit polyclonal, PM020 for immunocytochemistry), and histidine (rabbit polyclonal, PM032) (MBL, Woburn, MA); rabbit polyclonal antibody against SARM1 (NB100-94383; Novus Biologicals); antibodies against ubiquitin (mouse monoclonal, sc-8017), Tom20 (mouse monoclonal, sc-17764), Tom20 (rabbit polyclonal, sc-11415), and Bcl-2 (mouse monoclonal, sc-7382; Santa Cruz Biotechnology, Santa Cruz, CA); antibodies against ubiquitin (mouse monoclonal, 550944) and cytochrome c (mouse monoclonal, 556433; BD PharMingen, San Jose, CA); antibodies against tubulin (mouse monoclonal, T5168) and HA-agarose (mouse monoclonal, A2095 for immunoprecipitation; Sigma-Aldrich); and HRP-labeled TrueBlot anti-mouse IgG (18-8817-33; eBioscience, San Diego, CA).

Techniques: Transfection, Construct, Expressing, Control, Cotransfection, Knockdown, Over Expression

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: Primer sequences used in qRT-PCR analysis.

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: cDNA Synthesis

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: (A) Microscopic images of HK-2 cells of OGD/R model. Left, control cells cultured in normoxia condition; middle, cells cultured in 12 hours of hypoxia and 12 hours reoxygenation; right, cells cultured in 24 hours of hypoxia but no reoxygenation. (scale bar = 200μm) (B) Viability of cells in (A) detected via CCK8. (C) Western Blot of apoptosis related proteins BCL2, Caspase-3 and BAX. (D-F) Quantification of the protein level in (C) by ImageJ software for (D) BCL2, (E) Caspase-3, and (F) BAX. (G) mRNA level of YTHDF2 in HK-2 cells of OGD/R detected by RT-PCR. (H, I) YTHDF2 protein detected in HK-2 cells of OGD/R by (H)Western Blot and (I) quantified base on the gray value of bands. Data was expressed as the mean ± SD of three repeated experiments. **P < 0.01, ***P < 0.001 and ****P < 0.0001 vs. the control group. (n = 3 for cell experiments).

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: Control, Cell Culture, Western Blot, Software, Reverse Transcription Polymerase Chain Reaction

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: (A) Diagram showing the procedure of IRI model in mouse kidney. (B, C) Morphological changes were visualized in kidney with (B) H&E staining and then (C) quantified. (B) Representative images of control (upper left) and modeled tissues (lower left) were compared in 40 × magnification (Bar = 200μm). Renal tubules with regular morphology (upper right) or lumen expansion (lower right) were further magnified from left images (Bar = 50μm). (C) Frequency of enlarged lumens was counted within control and modeled renal cortex. For either group, 5 fields of vision were picked randomly from 5 kidneys. The result was presented as percentage of area occupied by tubules with lumen expansion in fields of view. (D, E) Microscopic imaging of basal membrane of renal tubules via (D) PASM staining and (E) quantified within cortex area. (D) Representative images of control (upper left) and modeled tissues (upper right) were compared in 40 × magnification (Bar = 200μm). Renal tubules with (lower left) or without (lower right) PASM staining were further magnified from upper images (Bar = 50μm). (E) Frequency of lumens absent of PASM was counted within renal cortex under 40 × magnification. For either group, 5 fields of vision were picked randomly from 5 kidneys. The result was presented as percentage of area occupied by tubules negative of PASM. (F) mRNA level of YTHDF2 in control and IRI kidneys by RT-PCR. (G, H) YTHDF2 protein level in control and IRI kidneys detected by (G) Western Blot and (H) quantified results based the gray value of bands. (I, J) Immunofluorescent imaging of YTHDF2 protein in (I) normal or IRI renal tissue under 60 × magnification (Bar = 20μm), while the staining intensity of was (J) quantified in 5 random fields of view for both groups. Data was expressed as the mean ± SD of three repeated experiments. * P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (n = 5 for mouse experiments).

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: Staining, Control, Imaging, Membrane, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: (A) Microscopic images of HK-2 cells infected with lentivirus for the ectopic expression of YTHDF2 in bright field (left) and fluorescent field (right). The fluorescence signal was generated by the GFP protein in the lentiviral vector. (scale bar = 200μm) (B-C) Confirmation of enhanced YTHDF2 protein level with (B) Western Blot and (C) the quantified results. (D) Morphological characterization of YTHDF2 overexpressed and control cells in OGD/R model. (scale bar = 200μm) (E) Viability of cells with/without YTHDF2 overexpression in OGD/R model. (F-I) The trend of apoptosis related proteins indicted with (F) Western Blot upon YTHDF2 overexpression in OGD/R model and quantified respectively for (G) BCL2, (H) BAX and (I) Caspase-3. Data was expressed as the mean ± SD of three repeated experiments. * P < 0.05, **P < 0.01 and ****P < 0.0001.

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: Infection, Expressing, Fluorescence, Generated, Plasmid Preparation, Western Blot, Control, Over Expression

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: (A) Microscopic images of HK-2 cells infected with lentivirus for the knockdown of YTHDF2 in bright field (left) and fluorescent field (right). The fluorescence signal was generated by the GFP protein in the lentiviral vector. (scale bar = 200μm) (B-C) Confirmation of impaired YTHDF2 protein level with (B) Western Blot and (C) the quantified results. (D) Morphological characterization of YTHDF2 knockdown and control cells in OGD/R model. (scale bar = 200μm) (E) Viability of cells with/without YTHDF2 knockdown in OGD/R model. Data was expressed as the mean ± SD of three repeated experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: Infection, Knockdown, Fluorescence, Generated, Plasmid Preparation, Western Blot, Control

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: (A-B) The mRNA level of TUG1 detected by RT-PCR in HK-2 cells with (A) YTHDF2 overexpression and (B) YTHDF2 knockdown under hypoxia/reoxygenation condition. (C) RNA immunoprecipitation assay demonstrated YTHDF2 binding to TUG1 (****P < 0.0001). (D) The candidate m 6 A sites on TUG1 RNA predicted with SRAMP. (E) groups of m 6 A sites defined with their confidence level scored by SRAMP. (F) Potential binding sites of YTHDF2 recognized on TUG1 RNA chain based on the defined motif (A/G/U)GAC(A/U) (G) Number of candidate sites matching both criteria, potential of m 6 A modification and allowance for YTHDF2 binding. (H)The expression of miR-449b-5p in OGD/R-induced HK-2 cells with YTHDF2 overexpression were tested using qRT-PCR. (I-J) MMP2 proteins indicted with (I) Western Blot upon YTHDF2 overexpression in OGD/R model and (J) quantified respectively for MMP2. Data was expressed as the mean ± SD of three repeated experiments. * P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: Reverse Transcription Polymerase Chain Reaction, Over Expression, Knockdown, RNA Immunoprecipitation, Binding Assay, Modification, Expressing, Quantitative RT-PCR, Western Blot

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: 21 potential binding sites of YTHDF2 protein on TUG1 RNA.

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: Binding Assay, Sequencing

Journal: Oncology Letters

Article Title: Overexpression of close homolog of L1 enhances the chemosensitivity of lung cancer cells via inhibition of the Akt pathway

doi: 10.3892/ol.2020.11972

Figure Lengend Snippet: CHL1 is downregulated in DDP and PTX-resistant A549 cells. (A) Cell survival of A549 and A549-resistant cells (A549/DDP and A549/PTX) treated with increasing concentrations of DDP and PTX, as assessed by MTT assay. (B) The IC 50 values of DDP in A549/DDP and A549 cells, and the IC 50 values of PTX in A549/PTX and A549 cells. *P<0.05 vs. A549 cells. (C) Western blotting demonstrated the expression of drug resistance-related proteins MDR1, MRP and LRP in A549 cells and A549-resistant cells (A549/DDP and A549/PTX). *P<0.05 vs. A549 cells. The protein and mRNA expression levels of CHL1 in A549 cells and A549-resistant cells (A549/DDP and A549/PTX) were analysed by (D) western blotting and (E) reverse transcription-quantitative PCR, respectively. *P<0.05 vs. A549 cells. (F) The mRNA expression of CHL1 in H460 and H460/DDP cells in the GSE21656 dataset. *P<0.05 vs. H460 cells. CHL1, close homolog of L1; DDP, cisplatin; PTX, paclitaxel; MDR1, multi-drug resistance gene 1; MRP, multidrug resistance-associated protein; LRP, low-density lipoprotein receptor-related protein; IC50, half maximal inhibitory concentration.

Article Snippet: Next, the proteins were transferred onto a polyvinylidene membrane (Thermo Fisher Scientific, Inc.), blocked with 5% BSA (Thermo Fisher Scientific, Inc.) for 2 h at 4°C, and incubated overnight at 4°C with primary antibodies against CHL1 (1:500; cat. no. 25250-1-AP; ProteinTech, Inc.), multi-drug resistance gene 1 (MDR1; 1:500; cat. no. 22336-1-AP; ProteinTech, Inc.), multidrug resistance-associated protein (MRP; 1:500; cat. no. 67228-1-Ig; ProteinTech, Inc.), low-density lipoprotein receptor-related protein (LRP; 1:500; cat. no. 22336-1-AP; ProteinTech, Inc.), phosphorylated (p)-Akt (1:1,000; cat. no. ab38449; Abcam,) and Akt (1:2,000; cat. no. ab227385; Abcam).

Techniques: MTT Assay, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Concentration Assay