Journal: bioRxiv

Article Title: Targeted protein degradation in lysosome utilizing naturally produced bifunctional antibodies with high levels of mannose 6-phosphate glycans

doi: 10.1101/2024.09.03.611037

Figure Lengend Snippet: A . Schematic showing the procedure for detection of FLAG-mCherry and NCA-FLAG or PNCA-FLAG complexes binding toward CI-MPR. B . Bound mCherry fluorescence from (A) for NCA-FLAG (gray) or PNCA-FLAG (red). N=3 or 4. C . HepG2 cells were incubated with FLAG-mCherry and NCA-FLAG or PNCA-FLAG overnight with or without 2 mM M6P and cells were examined by fluorescent microscopy to detect mCherry signal (red). Lysosomes were stained with LysoTracker (green) and Hoechst (blue) for nuclei. D-H . Cell lines (D. HEK293T, E. Tay-Sachs Patient Fibroblasts, F. Daoy, G. HepG2, or H. MDA-MB-231) were incubated with FLAG-HexM enzyme with NCA-FLAG or PNCA-FLAG or with addition of 2 mM M6P. HexM internalization was determined by performing a HexM activity assay and activity graphed – HexM alone (gray), NCA-FLAG with HexM (Green), PNCA-FLAG with HexM (Blue), M6P added (black striped bars). Background HexM activity in untreated group wa subtracted. Dot represents each independent experiment. N=3-6. I . HEK293T CI-MPR -/- cells were generated by CRISPR-Cas9. Western blot shows CI-MPR deficiency in the cells. J . Graphs depict internalized HexM activity in CI-MPR -/- cells. Dot represents each independent experiment. N=3. Data are represented as mean values; error bars represent the standard deviation of biological replicates.

Article Snippet: HepG2 (ATCC, HB-8065), Daoy (ATCC, HTB-186) and A431 (Sigma, 85090402) cells were cultured in MEM medium (Corning, 10-009-CV) with 10% FBS.

Techniques: Binding Assay, Fluorescence, Incubation, Microscopy, Staining, Activity Assay, Generated, CRISPR, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Targeted protein degradation in lysosome utilizing naturally produced bifunctional antibodies with high levels of mannose 6-phosphate glycans

doi: 10.1101/2024.09.03.611037

Figure Lengend Snippet: A . Schematic demonstrating production of Ab-TNFα and PNCA-TNFα in CHO cells. B . Western blotting to examine the Ab-TNFα and PNCA-TNFα heavy chain N-glycosylation after Endo-H and PNGase-F treatment. C-D . CI-MPR affinity chromatograph to determine the binding of Ab-TNFα and PNCA-TNFα. To visualize the binding signal, Alexa Fluor 594 conjugated to Ab-TNFα (C) or PNCA-TNFα (D) was used for the assay. Red dash line indicates the concentration of free M6P for antibody elution from the CI-MPR column. E . Schematic depicting internalization and degradation of extracellular TNFα b PNCA-TNFα through CI-MPR to the lysosome. F . HepG2 cells were incubated with mCherry-TNFα and Ab-TNFα or PNCA-TNFα overnight with or without 2 mM M6P and cells were examined by fluorescent microscopy to detect mCherry-TNFα signal (red). Lysosomes were stained with LysoTracker (green) and Hoechst for Nuclei (blue). G . Internalized mCherry-TNFα protein was analyzed by western blot for cell lysates using antibodies to detect mCherry, TNFα, and GAPDH as a loading control. H . Western blot analysis for internalized mCherry-TNFα with the addition of 1% or 0.5% of lysosomal protease inhibitors (PI). Antibodies are used to detect mCherry, TNFα and GAPDH, as a loading control.

Article Snippet: HepG2 (ATCC, HB-8065), Daoy (ATCC, HTB-186) and A431 (Sigma, 85090402) cells were cultured in MEM medium (Corning, 10-009-CV) with 10% FBS.

Techniques: Western Blot, Glycoproteomics, Binding Assay, Concentration Assay, Incubation, Microscopy, Staining, Control

Journal: The Journal of biological chemistry

Article Title: A minimally lipidated form of cell-derived apolipoprotein E exhibits isoform-specific stimulation of neurite outgrowth in the absence of exogenous lipids or lipoproteins.

doi: 10.1074/jbc.273.7.4206

Figure Lengend Snippet: FIG. 2. Western blotting of apoE secreted by stably transfected cell lines. Conditioned media (100 mg of protein) were separated by SDS-10% polyacrylamide gel electrophoresis and were electroblotted to a nitrocellulose membrane. The membrane was probed with an affinity purified polyclonal goat anti-human apoE antibody. After incubation with a horseradish peroxidase-conjugated anti-goat IgG, bands were visualized by enhanced chemiluminescence. The slight difference in mobility of the apoE3 and apoE4 bands is due to curvature in the running front and was not seen in other experiments.

Article Snippet: The cells were washed three times with PBS containing 0.5% Triton X-100 and incubated with a rhodamine-conjugated donkey anti-goat IgG antibody (Jackson Immuno Research) at 1:2000 in in PBS containing 1% FBS and 0.5% Triton X-100 for 1 h at 37 °C.

Techniques: Western Blot, Stable Transfection, Transfection, Polyacrylamide Gel Electrophoresis, Membrane, Affinity Purification, Incubation

Journal: The Journal of biological chemistry

Article Title: A minimally lipidated form of cell-derived apolipoprotein E exhibits isoform-specific stimulation of neurite outgrowth in the absence of exogenous lipids or lipoproteins.

doi: 10.1074/jbc.273.7.4206

Figure Lengend Snippet: FIG. 3. Immunocytochemical detection of apoE in stably transfected cell lines. Neuro-2a parental cells (A and B), Neuro-2a apoE3- secreting cells (C), and Neuro-2a apoE4-secreting cells (D) were grown on glass coverslips. ApoE localization was detected with an affinity purified polyclonal goat anti-human apoE antibody and a rhodamine-conjugated secondary antibody (A, C, and D). The white arrows highlight the concentrated localization of apoE in the growth cone domains in the apoE isoform-expressing Neuro-2a cells. B is the transmitted light image of the parental Neuro-2a cells found in A (black arrows highlight 2 Neuro-2a cells). All images were collected by confocal microscopy with a 40 3 oil immersion lens.

Article Snippet: The cells were washed three times with PBS containing 0.5% Triton X-100 and incubated with a rhodamine-conjugated donkey anti-goat IgG antibody (Jackson Immuno Research) at 1:2000 in in PBS containing 1% FBS and 0.5% Triton X-100 for 1 h at 37 °C.

Techniques: Stable Transfection, Transfection, Affinity Purification, Expressing, Confocal Microscopy

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 2. Inhibition of p38 blocks NGF-induced neurite out- growth. A and B, PC12 cells were pretreated with the indicated con- centrations of SB203580 or 30 mM PD98059 for 30 min prior to treat- ment with 100 ng/ml NGF for 60 h. Representative images under a phase-contrast microscope (A) and quantitation of the percentage of cells with neurites (B) are shown. C and D, cells were cotransfected with pEGFP-C1 together with an empty expression vector SRa (2) or an expression vector encoding kinase-negative MKK6 (KN-MKK6), wild type p38 (WT-p38), or dominant-negative p38 (AGF-p38) (15). After 12 h the cells were treated with or without 10 mM SB203580. Then, 48 h after the transfection the cells were treated with or without 100 ng/ml NGF. Representative images of the transfected cells 60 h after NGF addition identified by the fluorescence of GFP (C) and quantitation of the percentage of cells with neurites (D) are shown.

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Microscopy, Quantitation Assay, Expressing, Plasmid Preparation, Dominant Negative Mutation, Transfection, Fluorescence

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 1. NGF induces p38 activation as well as ERK/MAPK ac- tivation. A, PC12 cells were treated with 100 ng/ml NGF or 50 mM arsenite for the indicated times (left) or with the indicated concentra- tions of NGF for 10 min (right), and the cell extracts were subjected to the immune complex kinase assay for p38 using activating transcrip- tion factor 2, ATF2, as a substrate (upper). The same cell extracts were subjected to immunoblotting with anti-phospho-p38 (middle) or anti- p38 antibodies (bottom). B, cells were pretreated with or without a p38 inhibitor SB203580 at 10 mM (lower or upper, respectively) for 30 min prior to NGF treatment as indicated, and the extracts were subjected to immunoblotting with anti-ERK/MAPK antibody. The electrophoreti- cally retarded bands represent active forms, i.e. phosphorylated forms of ERK/MAPK (ERK1 and ERK2, arrowheads) against inactive forms (arrows).

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Immune Complex Kinase Assay, Western Blot

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 3. Expression of a constitutively active MAPKK/MEK (SE- SE-KK) induces p38 activation as well as ERK/MAPK, and the p38 inhibitor blocks neurite outgrowth induced by SESE-KK. A, PC12 cells were cotransfected with pEGFP-C1 and either an empty expression vector SRa (2) or a constitutively active construct of MAPKK/MEK (SESE-KK (13); equivalent to Glu-217/Glu-221 MAPKK/ MEK in Ref. 7) expression vector and were treated with or without 10 mM SB203580. B, cells were cotransfected with HA-p38 or HA-ERK/ MAPK (MAPK) together with an empty expression vector SRa (2) or an expression vector encoding wild type MAPKK/MEK (WT-KK) or SE- SE-KK and assayed for the activity of HA-p38 or HA-MAPK. The activity of HA-p38 was also measured in cells treated with 100 ng/ml NGF for 10 min (1NGF). C, cells were cotransfected with pEGFP-C1 and either an empty expression vector SRa (2) or an SESE-KK expres- sion vector and were subjected to immunostaining with anti-phospho- p38 antibody.

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Activation Assay, Plasmid Preparation, Construct, Activity Assay, Immunostaining

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 4. EGF induces transient activation of p38 and, when combined with sustained activation of p38, causes neurite out- growth in PC12 cells. A, PC12 cells were treated with either 30 nM EGF (upper), 50 mM arsenite (upper), or 100 ng/ml NGF (lower) for the indicated times and assayed for p38 activity as described in the legend to Fig. 1A. B, cells were treated with EGF, NGF, or arsenite for the indicated times and subjected to immunostaining with anti-phospho- p38 antibody (lower; phase contrast, upper). C, cells were transfected with pEGFP-C1 together with either an empty expression vector SRa (2) or both wild type MKK6 and wild type p38 expression vectors (MKK6 & p38) and treated with or without 10 mM SB203580. 48 h after the transfection the cells were treated with or without EGF. Represent- ative images of the transfected cells 72 h after EGF addition identified by the fluorescence of GFP are shown. D, cells were treated with EGF, arsenite, or both for 1 h, washed, and then incubated in fresh medium. Representative images 60 h after the treatment under a phase-contrast microscope are shown.

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Activity Assay, Immunostaining, Transfection, Expressing, Plasmid Preparation, Fluorescence, Incubation, Microscopy

Journal: The Journal of biological chemistry

Article Title: A bipartite nuclear localization signal is required for p53 nuclear import regulated by a carboxyl-terminal domain.

doi: 10.1074/jbc.274.46.32699

Figure Lengend Snippet: FIG. 1. Effect of LMB on the subcellular localization of Lys305- mutated p53. MCF-7 cells grown on coverslips were transfected with p53K305-GFP and treated with or without 100 nM LMB for 4 h. The cellular localization of cyclin B1 in transfected cells was determined by immunostaining with a monoclonal anti-cyclin B1 antibody and an LRSC-conjugated goat anti-mouse IgG. The localizations of Lys305-mu- tated p53 and cyclin B1 were differentiated by GFP and LRSC fluores- cence using fluorescence microscopy.

Article Snippet: After washing, one-twentieth of the beads were removed to analyze the amount of immobilized GST fusion proteins by immunoblotting with the anti-p53 pAb122 hybridoma supernatant (ATCC TIB116) or the anti-GST mouse IgG1 monoclonal antibody (Santa Cruz Biotechnology).

Techniques: Transfection, Immunostaining, Fluorescence, Microscopy

Journal: The Journal of biological chemistry

Article Title: A bipartite nuclear localization signal is required for p53 nuclear import regulated by a carboxyl-terminal domain.

doi: 10.1074/jbc.274.46.32699

Figure Lengend Snippet: FIG. 2. Subcellular localizations of p53 nuclear localization signals and PK fusion derivatives. The p53 residues 316–322 (NLSI), 305–322, or K305N-322 were linked to the KpnI sites in the carboxyl terminus of a Myc-tagged PK cDNA. The different fusion constructs were transiently transfected into MCF-7 cells, and the cel- lular localization of fusion proteins was determined by immunostaining with a monoclonal anti-c-Myc antibody and a fluorescein isothiocya- nate-conjugated goat anti-mouse IgG.

Article Snippet: After washing, one-twentieth of the beads were removed to analyze the amount of immobilized GST fusion proteins by immunoblotting with the anti-p53 pAb122 hybridoma supernatant (ATCC TIB116) or the anti-GST mouse IgG1 monoclonal antibody (Santa Cruz Biotechnology).

Techniques: Construct, Transfection, Immunostaining

Journal: The Journal of biological chemistry

Article Title: A bipartite nuclear localization signal is required for p53 nuclear import regulated by a carboxyl-terminal domain.

doi: 10.1074/jbc.274.46.32699

Figure Lengend Snippet: FIG. 5. Co-localization of the p53 oligomerization domain with wild-type and Lys305-mutated p53. A FLAG fusion protein contain- ing the p53 oligomerization domain (residues 325–369) was expressed alone or co-expressed with p53-GFP or p53K305N-GFP in MCF-7 cells. The cellular localization of FLAG-p53-(325–369) was determined by immunostaining with a monoclonal anti-FLAG antibody and an LRSC- conjugated goat anti-mouse IgG.

Article Snippet: After washing, one-twentieth of the beads were removed to analyze the amount of immobilized GST fusion proteins by immunoblotting with the anti-p53 pAb122 hybridoma supernatant (ATCC TIB116) or the anti-GST mouse IgG1 monoclonal antibody (Santa Cruz Biotechnology).

Techniques: Immunostaining

Journal: Nature

Article Title: DAXX represents a new type of protein-folding enabler.

doi: 10.1038/s41586-021-03824-5

Figure Lengend Snippet: Fig. 1 | DAXX prevents protein misfolding and aggregation. a, b, Heat-induced luciferase inactivation (a, 5 nM) and aggregation (b, 200 nM) in presence or absence of GST, DAXX and HSP70–HSP40 at 200 nM (a) or at the indicated molar ratios (b). c, Aggregation of ATXN1(82Q) (50 nM) in the presence or absence of glutathione S-transferase (GST) or DAXX (200 nM each). Raw data for this and other gels are found in Supplementary Fig 1. PE, pelletable aggregates that were soluble with SDS; SN, supernatant. d–f, Fibrillization of α-Syn (70 μM) in the presence GST, HSP70–HSP40, HSPs (HSP70–HSP40–HSP104(A503S)) (200 nM each) and DAXX (100–400 nM) was assayed by ThT-binding (d), electron microscopy (e; red arrows, fibrils; blue arrows, large oligomers; scale bar, 100 nm), and sedimentation followed by dot blot for SDS-soluble and SDS-resistant (SR) aggregates and total α-Syn and by disuccinimidyl suberate (DSS) cross-linking for soluble oligomers (f). IB, immunoblot. g–i, Fibrillization of Aβ42 monomers (10 μM) in the absence or presence of DAXX (50–600 nM) (g, h), and viability of SH-SY5Y cells treated with Aβ42 pre-incubated with or without DAXX (i). An ATP-regeneration system was included with heat-shock proteins but not DAXX (all subsequent experiments with heat-shock proteins, but not experiments with DAXX, also contained an ATP-regeneration system). Data are mean or mean ± s.d. (n = 4 for i, and 3 for the rest) and are representative of three independent experiments. *P < 0.05, NS, not significant; unpaired Student’s t-test.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Antibodies against the following proteins/epitopes were purchased from the indicated sources: GAPDH (sc-47724,Mouse, WB, IP, IF and IHC(P)), His (sc-8036, Mouse, IP, WB, IHC(P), ELISA, IF, FCM), GST (sc-138, Mouse, IP, WB, IHC(P), ELISA, IF, FCM), p53DO1 (sc-126, Mouse, WB, IP, IF, IHC(P) and FCM), Mdm2 (sc-965, Mouse, WB, IP, IF and IHC(P)), DAXX (sc-8043, Mouse IP,WB, IHC(P), ELISA, IF, FCM), α-synuclein syn211 (sc-12767, Mouse, IP, WB, IHC(P), ELISA, IF, and FCM) (Santa Cruz Biotechnology); Flag (#14793, Rabbit, WB, IP, IHC, ChIP, IF, FCM, ELISA) and DAXX (#4533S, Rabbit, WB, IP, IHC, ChIP, IF, FCM, ELISA) (Cell Signaling Technology); p53 (PAb1620, #OP33, Mouse, WB, IP, IF; PAb240, #OP29, Mouse, WB, IP), and Mdm2 (#OP46, Mouse, WB, IP) (Calbiochem); and HA (ab137838, Rabbit, WB, ICC/IF, IP) and Luciferase (ab21176, Rabbit, ICC/IF, WB) (Abcam); βAmyloid 1-42 (#805509) (BioLegend, Mouse, WB, IHC(P), ELISA).

Techniques: Luciferase, Binding Assay, Electron Microscopy, Sedimentation, Dot Blot, Western Blot, Incubation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The natural product vioprolide A exerts anti-inflammatory actions through inhibition of its cellular target NOP14 and downregulation of importin-dependent NF-ĸB p65 nuclear translocation.

doi: 10.1016/j.biopha.2021.112255

Figure Lengend Snippet: Fig. 7. Vioprolide A reduces importin subunit alpha-1 total protein level and influences importin subunit alpha-1/beta-1 nuclear localization. a-c Confluent HUVECs were treated with vioprolide A (10 nM) as indicated. Total protein levels of importin subunit alpha-1 (a), importin subunit alpha-3 (b) and importin subunit beta-1 (c) were determined by western blot analysis. One representative blot out of 3 is shown. d-f Confluent HUVECs were treated with vioprolide A (10 nM) for 16 h followed by activation with TNF (10 ng/ml) as indicated. The nuclear fractions were separated and total nuclear protein expression of importin subunit alpha-1 (d), importin subunit beta-1 (e) and importin subunit alpha-3 (f) was determined by western blot analysis. One representative blot out of 3 is shown. VioA, vioprolide A; Imp, importin; Topo1, topoisomerase 1. Data are expressed as mean ± SEM. n = 3. *P ≤0.05 vs. negative control (a-c) or TNF control (d-f). #P ≤0.05 vs. negative control (d-f).

Article Snippet: Unspecific binding sites were blocked with 0.2% BSA (MilliporeSigma) for 30 min and cells were treated with primary antibody for 2 h. The following primary antibodies were used: rabbit anti-human NF-ĸB p65 (1:400, sc8008; Santa Cruz Biotechnology) and rabbit anti-human importin subunit alpha-1 (1:400, #14372; Cell Signaling/New England Biolabs).

Techniques: Western Blot, Activation Assay, Expressing, Negative Control, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The natural product vioprolide A exerts anti-inflammatory actions through inhibition of its cellular target NOP14 and downregulation of importin-dependent NF-ĸB p65 nuclear translocation.

doi: 10.1016/j.biopha.2021.112255

Figure Lengend Snippet: Fig. 8. NOP14 knockdown influences TNF- activated inflammatory processes in HUVECs. a Confluent HUVECs were treated with 300 nM vioprolide A for 1 h or left untreated. Cells were exposed to increasing temperatures between 42.3 and 57.8 ◦C and lysed by repeated freeze- thaw cycles in liquid nitrogen. Thermal aggre gation curves of NOP14 were analyzed by western blot. One representative blot out of 4 is shown. b-e Subconfluent cells were transfected with siRNA against NOP14 (siNOP14, 60 nM) or non-targeting siRNA (nt siRNA, 60 nM) as control using GeneTrans II Transfection Re agent. 30 h after transfection, HUVECs were treated with 10 nM vioprolide A for 16 h. b HUVECs were activated with TNF (10 ng/ml) for 6 h and fluorescence-labeled THP-1 cells were allowed to adhere to the HUVEC mono layer for 5 min. The amount of adhered THP-1 was detected by fluorescence measurement. c HUVECs were activated with TNF (10 ng/ml) for 4 h. The mRNA expression of ICAM1 was analyzed by quantitative PCR. d HUVECs were activated with TNF (10 ng/ml) for 30 min p65 was visualized by immunocytochemistry and fluorescence microscopy. One representative experiment out of 3 is shown. Scale bar, 200 µm. e Importin subunit alpha-1 was visu alized by immunocytochemistry and fluores cence microscopy. One representative experiment out of 3 is shown. Scale bar, 50 µm. Tagg, aggregation temperature; VioA, vioprolide A. Data are expressed as mean ± SEM. n = 4 (a), n = 5 (b), n = 3 (c-e). *P ≤0.05 vs. TNF control.

Article Snippet: Unspecific binding sites were blocked with 0.2% BSA (MilliporeSigma) for 30 min and cells were treated with primary antibody for 2 h. The following primary antibodies were used: rabbit anti-human NF-ĸB p65 (1:400, sc8008; Santa Cruz Biotechnology) and rabbit anti-human importin subunit alpha-1 (1:400, #14372; Cell Signaling/New England Biolabs).

Techniques: Knockdown, Western Blot, Transfection, Control, Fluorescence, Labeling, Expressing, Real-time Polymerase Chain Reaction, Immunocytochemistry, Microscopy

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Pantoprazole pretreatment elevates sensitivity to vincristine in drug-resistant oral epidermoid carcinoma in vitro and in vivo.

doi: 10.1016/j.biopha.2019.109478

Figure Lengend Snippet: Fig. 6. PPZ treatment induces an increase in lysosomal pH and inhibits the activity of lysosomal enzyme acid phosphatase in KB/V cells. KB/V cells were treated with various concentrations of PPZ (a) or V-ATPase inhibitor Baf-A1 (b) for 4–24 h, and then the supernatant was harvested for pHe measurements. c Effect of PPZ on lysosomal pH in KB/V cells was evaluated by LysoSensor Green probe using fluorescence microscope. d The activity of lysosomal enzyme acid phosphatase in KB/V cells treated with various concentrations of PPZ for 24 h. e Effect of PPZ on the expression of V-ATPase in KB/V cells was detected and quantified using Western blotting analysis. For a, b, d and e, data are presented as the mean ± SD of three separate experiments. *P < 0.05, **P < 0.01 vs. KB/V control group. f Effect of V-ATPase inhibitor Baf-A1 on the synergistic cytotoxicity of PPZ and VCR in KB/V cells. KB/V cells were treated with Baf-A1 (50, 200 nM) with or without PPZ and VCR, then the cell viability was detected by MTT assay. Data are presented as the mean ± SD from three separate experiments. *P < 0.05, **P < 0.01 vs. KB/V control group; △△P < 0.01 vs. PPZ alone group; ##P < 0.01 vs. PPZ and VCR combination group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Article Snippet: Membranes were blocked in 5% (w/v) non-fat milk before incubation with primary antibodies (1:1000 in primary antibody diluent) against PARP (#9532), cleavedcaspase 3 (#9664), Bcl-xL(#2762), Bcl-2 (#2872), Bax (#2772), p21 (#2947), Cyclin B1 (#4138), cdc2 (#9112), p-cdc2 (#9111), MMP2 (#4022), MMP9 (#3852), P-gp (#13342), EGFR (#2232), p-EGFR (#2235), p-ERK (#4370), p-p38 (#4511), PI3K (#3358), Akt (#2965), p-Akt (#2965), mTOR (#2972), p-mTOR (#2971), β-actin (#4970) (all from Cell Signaling Technology, CST, Boston, MA) and V-ATPase (14418-1-AP, Proteintech, Wuhan, China) at 4 °C overnight.

Techniques: Activity Assay, Microscopy, Expressing, Western Blot, Control, MTT Assay

Journal: The Journal of cell biology

Article Title: ARFRP1 functions upstream of ARL1 and ARL5 to coordinate recruitment of distinct tethering factors to the trans-Golgi network.

doi: 10.1083/jcb.201905097

Figure Lengend Snippet: Figure 2. Both ARL5 and ARFRP1 are required for localization of GARP to the TGN. (A) KO of TGN-localized small GTPases in HeLa cells confirmed by immunoblot analysis with antibodies to the proteins indicated on the right. In this figure and subsequent figures, ARL5 KO represents KO of both ARL5A and ARL5B, and RAB6 KO represents KO of both RAB6A and RAB6B. α-Tubulin was used as a loading control. The positions of molecular mass markers are indicated on the left. (B) Immunofluorescence microscopy of WT and KO HeLa cells transfected with a plasmid encoding VPS54-13Myc and stained for the Myc epitope (red), giantin (green), and nuclei (DAPI; blue). Scale bars: 10 μm. Insets are magnified views of the boxed areas. Inset scale bars: 5 μm. (C) Quantification of the percentage of cells exhibiting VPS54-13Myc staining at the TGN. Values are the mean ± SEM from three independent experiments. More than 100 cells per sample were counted in each experiment. The statistical significance of the differences relative to WT cells was determined using Dunnett’s test. **, P < 0.01; ***, P < 0.001.

Article Snippet: The following antibodies were used for immunoblotting and/or immunofluorescence microscopy: rabbit anti-VPS51 (HPA039650; Atlas Antibodies), rabbit anti-VPS52 made in our laboratory (Pérez-Victoria et al., 2008), rabbit anti-VPS53 (HPA024446; Atlas Antibodies), mouse anti-VPS50 (FLJ20097, monoclonal antibody M01, 2D11; Abnova), mouse anti-Myc epitope (9E10; Santa Cruz Biotechnology), sheep anti-TGN46 (AHP500G; Bio-Rad), mouse anti–β-actin (G043; Applied Biological Materials), rabbit antigiantin (ab80864; Abcam), mouse HRP-conjugated anti-α-tubulin (DM1A; Santa Cruz Biotechnology), rabbit anti-ARL1 (16012-1-AP; Proteintech), rabbit anti-ARFRP1 (PA5-50606; Thermo Fisher Scientific), mouse anti-ARL5A (sc-514680; Santa Cruz Biotechnology), rabbit anti-RAB6A (GTX110646; GeneTex), rabbit anti-GCC88 (HPA021323; Sigma-Aldrich), rabbit anti-GCC185 (HPA035849; Sigma-Aldrich), mouse anti-Golgin-245 (611281; BD Biosciences), mouse anti-GM130 (610822; BD Biosciences), mouse anti–Golgin-97 (A-21270; Thermo Fisher Scientific), rabbit anti-TMF1 (HPA008729, Sigma-Aldrich), monoclonal HRP-conjugated anti-GFP (Miltenyi Biotec Inc.), rabbit anti-GFP (A-11122; Thermo Fisher Scientific), HRP-conjugated goat anti-rabbit and donkey anti-mouse antibodies (Jackson ImmunoResearch), HRP-conjugated donkey antisheep (R&D Systems), and Alexa Fluor–conjugated secondary antibodies for immunostaining (Thermo Fisher Scientific).

Techniques: Western Blot, Control, Immunofluorescence, Microscopy, Transfection, Plasmid Preparation, Staining

Journal: The Journal of cell biology

Article Title: ARFRP1 functions upstream of ARL1 and ARL5 to coordinate recruitment of distinct tethering factors to the trans-Golgi network.

doi: 10.1083/jcb.201905097

Figure Lengend Snippet: Figure 5. Small GTPases required for localization of golgins to the TGN. (A) Immunofluorescence microscopy of WT, ARL1-KO, ARFRP1-KO, ARL5-KO, and RAB6-KO cells immunostained for endogenous Golgin-245, Golgin-97, GCC88, GCC185, or TMF1 and counterstained with DAPI (blue). Scale bars: 10 μm. Insets are magnified views of the boxed areas. Inset scale bars: 5 μm. Notice that ARL1 KO or ARFRP1 KO caused complete disappearance of Golgin-245 and GCC88, and a partial decrease in the intensity of Golgin-97, at the TGN; quantification in 10 cells per sample in three independent experiment showed that Golgin-97 decrease was 75.6% ± 2.4% in ARL1-KO cells and 55.0% ± 1.9% in ARFRP1-KO cells. (B) SDS-PAGE and immunoblot analysis of endogenous golgins and α-tubulin (loading control) in WT and KO cells. The positions of molecular mass markers are indicated on the left. (C) Immunofluorescence microscopy of RAB6- KO cells transfected with a plasmid encoding GFP-tagged mouse Rab6A (green), immunostained for endogenous GCC185 and TMF1 (red), and counterstained with DAPI (blue). Cells were examined for GFP fluorescence by confocal microscopy. Scale bars: 10 μm.

Article Snippet: The following antibodies were used for immunoblotting and/or immunofluorescence microscopy: rabbit anti-VPS51 (HPA039650; Atlas Antibodies), rabbit anti-VPS52 made in our laboratory (Pérez-Victoria et al., 2008), rabbit anti-VPS53 (HPA024446; Atlas Antibodies), mouse anti-VPS50 (FLJ20097, monoclonal antibody M01, 2D11; Abnova), mouse anti-Myc epitope (9E10; Santa Cruz Biotechnology), sheep anti-TGN46 (AHP500G; Bio-Rad), mouse anti–β-actin (G043; Applied Biological Materials), rabbit antigiantin (ab80864; Abcam), mouse HRP-conjugated anti-α-tubulin (DM1A; Santa Cruz Biotechnology), rabbit anti-ARL1 (16012-1-AP; Proteintech), rabbit anti-ARFRP1 (PA5-50606; Thermo Fisher Scientific), mouse anti-ARL5A (sc-514680; Santa Cruz Biotechnology), rabbit anti-RAB6A (GTX110646; GeneTex), rabbit anti-GCC88 (HPA021323; Sigma-Aldrich), rabbit anti-GCC185 (HPA035849; Sigma-Aldrich), mouse anti-Golgin-245 (611281; BD Biosciences), mouse anti-GM130 (610822; BD Biosciences), mouse anti–Golgin-97 (A-21270; Thermo Fisher Scientific), rabbit anti-TMF1 (HPA008729, Sigma-Aldrich), monoclonal HRP-conjugated anti-GFP (Miltenyi Biotec Inc.), rabbit anti-GFP (A-11122; Thermo Fisher Scientific), HRP-conjugated goat anti-rabbit and donkey anti-mouse antibodies (Jackson ImmunoResearch), HRP-conjugated donkey antisheep (R&D Systems), and Alexa Fluor–conjugated secondary antibodies for immunostaining (Thermo Fisher Scientific).

Techniques: Immunofluorescence, Microscopy, SDS Page, Western Blot, Control, Transfection, Plasmid Preparation, Fluorescence, Confocal Microscopy

Journal: Molecular plant

Article Title: DET1-mediated COP1 regulation avoids HY5 activity over second-site gene targets to tune plant photomorphogenesis.

doi: 10.1016/j.molp.2021.03.009

Figure Lengend Snippet: Figure 1. DET1- and COP1-associated proteins. (A) Schematic representation of proteins found to associate with DET1 and COP1 in TAP assays. Color code represents the maximum number of peptides for each represented protein found in a TAP assay as detailed in Supplemental Table 1. DET1 and COP1 proteins were expressed in Arabidopsis cell cultures. Five independent TAP experiments were performed for DET1 and two for COP1 (Supplemental Table 2). (B) MBP-COP1 and MBP-HY5 recombinant proteins expressed in E. coli pulled-down MYC-DET1 from 7-day-old Arabidopsis seedlings. MBP re- combinant protein was used as a control. Anti-MYC and anti-MBP antibodies were used for the immunoblots. (C and D) F€orster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET–FLIM) analysis of the interaction between COP1 or HY5 fused to GFP and DET1 (C), and HY5 or COP1 fused to RFP (D). Box plots show the distribution of 5–9 measurements ±SD. (E) FRET–FLIM analysis of the interaction between GFP-COP1 and RFP-HY5 upon cluc-DET1 co-expression. Box plots show the distribution of 10 measurements ±SD. All FRET assays were performed following transient expression in N. benthamiana leaves. Free RFP was used as negative control. FE, FRET efficiency. Asterisks indicate statistically significant differences according to Student’s t-test (****p < 0.0001; ***p < 0.001; *p < 0.01). For all FRET experiments three independent biological replicates were performed, and results from one replicate are shown.

Article Snippet: Pull-down assays MBP recombinant protein fusions were expressed in the Escherichia coli BL21 (DE3) strain carrying the corresponding coding sequence cloned into the pKM596 plasmid, a gift from David Waugh (Addgene plasmid #8837).

Techniques: Recombinant, Control, Western Blot, Förster Resonance Energy Transfer, Imaging, Microscopy, Expressing, Negative Control

Journal: Ecotoxicology and environmental safety

Article Title: Ameliorative effect of betulinic acid against zearalenone exposure triggers testicular dysfunction and oxidative stress in mice via p38/ERK MAPK inhibition and Nrf2-mediated antioxidant defense activation.

doi: 10.1016/j.ecoenv.2022.113561

Figure Lengend Snippet: Fig. 2. BA protected the aggravation of male reproduction injury in ZEA-induced mice. The morphology of the sperm was photographed using an optical microscope (A). The sperm motility was used to evaluate the male repro duction of mice, including sperm survival rate (B), sperm malformation rate (C), and sperm mortality rate (D). Protein and mRNA levels of ERα in testis were detected by immunoblotting and RT-PCR analysis, respectively, and the pro tein and mRNA levels were normalized to β-actin (E-G). The content of testosterone in serum was detected by ELISA kit (H). The mRNA expression of CLDN11 (I), CDH2 (J), and Vim (K) were measured by RT-PCR. Mean ± SEM, *P < 0.05 and **P < 0.01 represented a significant differ ence compared to the control group, while #P < 0.05 and ##P < 0.01 represented a signifi cant difference compared to the ZEA group.

Article Snippet: Testosterone enzyme linked immunosorbent assay (ELISA) kit (CSB-E05101m) was obtained from Cusabio Biotech Co. Ltd. (Wuhan, China).

Techniques: Microscopy, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Control