Journal: Bioactive materials

Article Title: Calcium phosphate-based materials regulate osteoclast-mediated osseointegration.

doi: 10.1016/j.bioactmat.2021.05.003

Figure Lengend Snippet: Fig. 5. Effect of Ca/P ratio in CPC on RANKL-RANK signaling. (a) Immunofluorescence analysis of RANK expression (red) in osteoclasts on surface of CPC scaffold. (b) Concentration of RANK was analysis by ELISA. The concentration of RANK was measured after inducing BMMs for 1 days. (c) Affinity between RANKL and RANK was measured in BIAcore T200 system. Human-RANK anchored on the chip was applied to interact with the human-RANKL dissolved into the CPC extract. The vehicle means MEM-α medium. (d) 1.67CPC promoted RANKL-induced phosphorylation of NF-κB p65, and degradation of IκBα. (e) p-p65 and (f) IκBα intensity of Western blot bands was calculated with normalizing to β-actin. The untreated medium was treated as control. (g–l) Relative mRNA expression of osteoclast was measured after inducing BMMs for 7 days in CPC extract. BMMs cultured with untreated medium was treated as control. (*P ＜ 0.05, **P ＜ 0.01,***P ＜ 0.001).

Article Snippet: The concentration of RANK was measured by Mouse RANK ELISA Kit (Boster Biological Technology, China), according to the manufacturer’s instructions and the total protein concentration was measured by BCA Protein Assay Kit (Beyotime, China), which was used to normalize the expression of RANK.

Techniques: Immunofluorescence, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Control, Cell Culture

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: Structure–function analysis of BCL11A defines a region of BCL11A that interacts with NTHL1 and stimulates its enzymatic activity. ( A ) Diagrammatic representation of the BCL11A-XL and fragments tested in pull-down and DNA repair assays. ( B ) Diagrammatic representation of the 5,6-dihydrothymidine (DHT) cleavage assay using a fluorophore reporter probe. The assay was performed using 5 nM of NTHL1 and 20 nM of BCL11A-XL, in the presence of 50 nM of BSA. ( C ) Diagrammatic representation of the thymine glycol (Tg) cleavage assay using a radioactively labeled probe. The assay was performed using purified NTHL1 (10 nM) and the indicated amounts of BSA and/or BCL11A-XL. The reaction in lane 1 was performed with a probe that contains a normal thymidine base instead of thymine glycol. ( D ) Pull-down assays were performed using His-tagged BCL11A fragments and either GST or GST-NTHL1, followed by immunoblotting with anti-His antibodies. ( E ) DHT cleavage assays were performed using 20 nM of BCL11A fragments, in the presence of absence of NTHL1 (5 nM), as indicated. An additional assay was performed with 40 nM of BCL11A 160–520 . All samples included 50 nM of BSA; the sample labeled ‘BSA’ included an additional 20 nM BSA. ( F ) Thymine-glycol (Tg) cleavage assays were performed using radioactively end-labeled double-stranded oligonucleotides containing a Tg oxidized base, and 250 nM BSA. Where indicated, NTHL1 (2.5 nM), BSA or BCL11A 160–520 (15 nM) were included in the reaction. Production of a cleaved product in samples that have not been treated with NaOH (lanes 1–4) requires the glycosylase and AP/lyase activities of NTHL1. In the absence of NaOH, NTHL1 alone cleaves 9.7% of the substrate, whereas BCL11A 160–520 cleaves 49%. In the presence of NaOH, NTHL1 alone cleaves 20.3% of the substrate, whereas BCL11A 160–520 cleaves 43.8%. ( G ) Radioactively end-labeled double-stranded oligonucleotides containing a thymine glycol (Tg) base were incubated with the indicated amount of NTHL1, BSA and BCL11A 160–520 . After incubation at 37°C, 50 mM sodium borohydride was added. The reactions were incubated for another 15 min at 37°C. After termination of the reaction, the trapped complexes were separated from free DNA by 10% SDS-PAGE. ( H ) Pull-down assays were performed using the His-tagged BCL11A 161-366 peptide and either GST or GST-NTHL1 1-312 , GST-NTHL1 88-312 or GST-NTHL1 1-116 followed by immunoblotting with anti-His antibodies.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Activity Assay, Cleavage Assay, Labeling, Purification, Western Blot, Incubation, SDS Page

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: The DNA repair and proliferation defects caused by BCL11A knockdown are rescued by ectopic expression of the BCL11A 160–520 Fragment. ( A , B , E and F ) MDA-MB-231 cells were infected with lentiviral vectors as indicated: an empty vector, a vector expressing BCL11A shRNA, and a vector expressing BCL11A 160–520 . ( A ) Cells were exposed to 50 μM H 2 O 2 for 20 min and allowed to recover for the indicated time before carrying out single cell gel electrophoresis at pH >13. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( B ) Cells were exposed to 50 μM H 2 O 2 for 20 min and allowed to recover for the indicated time before carrying out single cell gel electrophoresis at pH 10 after treatment of cells with the Endo III DNA glycosylase. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01.; * P < 0.05; Student's t -test. ( C ) MDA-MB-231 cells stably carrying an empty vector or a vector expressing BCL11A 160–520 were transfected with either of two distinct BCL11A dicer RNAs or a control dicer RNA. Genomic DNA was purified and abasic sites were quantified using an aldehyde-reactive probe. Results are an average of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( D ) MDA-MB-231 cells were stably infected with a vector expressing BCL11A shRNA under the control of a tetracycline-inducible promoter, as well as an empty vector or a vector expressing BCL11A 160–520 . Cells were treated or not with doxycycline for 3 days before cell extracts were prepared and analyzed in a DNA repair assay using as a substrate double-stranded oligonucleotide containing a thymine glycol base (Tg). Upon incubation in the presence of 32 P-dTTP, NTHL1 present in the cell extract removes the thymine glycol and introduces a single-strand break; Pol β adds a radioactively labeled thymidine and ligase III seals the strand-break thereby generating a single-strand that migrates more slowly on a denaturing gel. The amount of repair completion (top band) for each sample was normalized to the value of ‘Vector no dox’. The enzymatic activity of the BCL11A knockdown (‘vector + dox’) is 55.7% whereas the activity of ‘BCL11A 160–520 no dox’ is 365% and ‘BCL11A 160–520 + dox’ is 361%. ( E ) Cells were treated with increasing amounts of H 2 O 2 and then submitted to a clonogenic assay. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( F ) MDA-MB-231 cells carrying BCL11A 160–520 or not were infected with lentiviruses expressing either control or BCL11A shRNA. β-Gal associated senescence was measured 5 days after infection. All values are normalized to the control shRNA value. Error bars represent standard error. Results are a representative of one of two different experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( G ) MDA-MB-231 cells were stably infected with a lentiviral vector expressing a BCL11A shRNA under the control of a doxycycline-inducible promoter and either an empty vector or a vector expressing BCL11A 160–520 . Doxycycline was added to the medium or not and 3 days later cell proliferation was measured by staining with CellTrace™ CFSE. CFSE was added to the medium and a portion of the population was fixed immediately as the ‘0’ generation. The remaining cells were allowed to proliferate for 3 days. Cells were fixed and analyzed by flow cytometry. Small peaks within the CFSE profiles represent successive generations, as indicated above the peaks. The proliferation index is the total number of divisions divided by the number of cells that went into division. The division index is the average number of cell divisions, taking into account the cells that never divided.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Expressing, Infection, Plasmid Preparation, shRNA, Single Cell Gel Electrophoresis, Stable Transfection, Transfection, Purification, Incubation, Labeling, Activity Assay, Clonogenic Assay, Staining, Flow Cytometry

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: BCL11A interacts with Pol β and stimulates its enzymatic activities. ( A ) Nuclear protein extracts (500 μg) from MDA-MB-231 cells were submitted to co-immunoprecipitation (IP) alternatively with Pol β or BCL11A antibodies and then immunoblotted (IB) with either anti-Polβ or anti-BCL11A antibody. Input (1%) was loaded as a protein expression control. ( B ) 293 cells were transfected with vectors expressing fusion proteins containing either the N-terminal or C-terminal portion of Intein, as indicated: BCL11A 160–520 -V5-IN4b with either IC2-Flag-LIG3, IC2-Flag-FOXN2 or IC2-Flag-Pol β. Whole cell extracts were submitted to immunoblotting analysis with the V5 and FLAG antibodies. ( C ) Pull-down assays were performed using His-tagged BCL11A fragments and either GST or GST-Pol β, followed by immunoblotting with anti-His antibodies. ( D ) Diagrammatic representation of the strand displacement assay using a fluorophore reporter probe. F: FAM fluorophore; Q: quencher. The strand displacement assay was performed using 2.5 nM of BCL11A and 15 nM of Pol β. 50 nM of BSA were added to each reaction. ( E ) Double-stranded oligonucleotides containing a uracil residue were incubated with UDG and APE1 to form a gapped substrate for Pol β. The DNA was then incubated with 32 P-dCTP in the presence of DNA Pol β and either BSA or BCL11A 160–520 . ( F ) Double-stranded oligonucleotides containing a uracil residue were labeled at the 5'-end and incubated with UDG and APE1 to form a gapped substrate for Pol β. The gapped probe was then incubated with all 4 dNTPs in the presence of DNA Pol β and either 25 nM BSA or 25 nM BCL11A 160–520 . ( G ) Double-stranded oligonucleotides containing a uracil residue, that were labeled at the 3′ end using Klenow and fluorescent CF 660R dCTP, were incubated with UDG and APE1 to produce a single-strand nick with a 5′-deoxyribose phosphate (dRP). This DNA substrate was incubated with Pol β in the presence of BSA or BCL11A 160–520 . NaBH4 was added to most samples, except in lane 9, to prevent the spontaneous conversion of dRP into P. From four independent pairs of assays, we calculated an average 1.8 fold (±0.3) stimulation of Pol β dRP-lyase activity by BCL11A 160–520 .

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Immunoprecipitation, Expressing, Transfection, Western Blot, Incubation, Labeling, Activity Assay

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: The impact of BCL11A knockdown on DNA repair, genomic DNA damage and clonogenic efficiency is confirmed in other triple-negative breast cancer cells. (A and B) The triple-negative breast cancer cell lines BT549, MDA-MB-468 and Hs578T were transfected with either of two distinct BCL11A dicer RNAs or a control dicer RNA. After 2 days, cell extracts were prepared and in parallel cells were submitted to a comet assay. ( A ) Cell extracts were prepared and analyzed in a DNA repair assay with a probe containing a thymine glycol, as described in Figure . ( B ) A fraction of the cells was submitted to single cell gel electrophoresis at pH >13. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( C ) Cells were stably infected with a lentiviral vector expressing a BCL11A shRNA under the control of a doxycycline-inducible promoter. Doxycycline was added to the medium or not and 3 days later cells were treated with the indicated concentrations (0, 20, 50) of H 2 O 2 and then submitted to a clonogenic assay. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Transfection, Single Cell Gel Electrophoresis, Stable Transfection, Infection, Plasmid Preparation, Expressing, shRNA, Clonogenic Assay

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: Proximity Biotinylation with NTHL1-BirA*. ( A ) Flow chart of manipulations for the BioID experiment. Following treatment with tetracycline and addition of biotin to the medium, cells were treated (2 Gy) or not (0 Gy) with ionizing radiation prior to cell lysis, affinity purification on streptavidin beads and mass spectrometry identification. ( B ) Immunoblotting analysis of Flp-In™ T-Rex™ 293 cells that were engineered to stably carry vectors that express BirA*-FLAG or NTHL1-BirA*-FLAG upon tetracycline induction. ( C ) Immunoblotting analysis with streptavidin confirming the expression of BirA* fusion proteins and successful biotinylation by BirA*. ( D ) The Venn diagram shows the number of NTHL1-BirA* target proteins identified from mass spectrometry after removing all candidates with BFDR above 0.2, and proteins identified in cells expressing BirA*-FLAG and NLS-BirA*-FLAG. 146 preys were identified in both unirradiated and irradiated cells, while 41 and 36 preys were respectively identified only in unirradiated or irradiated cells. ( E ) Nuclear protein extracts (500 μg) from MDA-MB-231cells were submitted to immunoprecipitation (IP) alternatively with NTHL1 or BCL11A antibodies and then immunoblotted (IB) with either anti-NTHL1 or anti-BCL11A antibody. The control lanes did not include a primary antibody while the input was loaded directly onto the gel. ( F ) 293 cells were transfected with vectors expressing BCL11A and NTHL1 fusion proteins containing the N-terminal or C-terminal portion of intein, respectively: BCL11A-V5-IN and IC2-FLAG-NTHL1. Whole cell extracts were submitted to immunoblotting analysis with the V5 and FLAG antibodies. The new band detected by both antibodies represents the recombined protein.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Lysis, Affinity Purification, Mass Spectrometry, Western Blot, Stable Transfection, Expressing, Irradiation, Immunoprecipitation, Transfection

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: BCL11A knockdown causes an increase in genomic DNA damage and a delay in the repair of oxidized bases and abasic sites. MDA-MB-231 cells were infected with lentiviral vectors expressing a BCL11A shRNA or a non-targeting sequence. Knockdown of BCL11A is ∼60%. ( A ) Cells were exposed to 50 μM H 2 O 2 for 20 min and allowed to recover for the indicated times before carrying out single cell gel electrophoresis at pH > 13. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three independent experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01.; * P < 0.05; Student's t -test. ( B ) Cells were exposed to 50 μM H 2 O 2 for 20 min and allowed to recover for the indicated time before carrying out single cell gel electrophoresis at pH 10 and pH 10 after treatment of cells with the Endo III DNA glycosylase. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three independent experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( C ) Cells stably carrying a lentivirus expressing an shRNA against BCL11A or an empty vector were treated or not with 50 μM H 2 O 2 . Genomic DNA was purified and abasic sites were quantified using an aldehyde-reactive probe. Results are an average of three independent experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01.; * P < 0.05; Student's t -test.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Infection, Expressing, shRNA, Sequencing, Single Cell Gel Electrophoresis, Stable Transfection, Plasmid Preparation, Purification

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: Ectopic Expression of BCL11A 160–520 Accelerates DNA Repair and Increases Resistance to H 2 O 2 . ( A ) RPE1 cells expressing the BCL11A 160–520 peptide were exposed to 100 μM H 2 O 2 for 20 min and allowed to recover for the indicated time before carrying out single cell gel electrophoresis at pH >13. Comet tail moments were scored for at least 100 cells per condition. Results are a representative of one of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01.; * P < 0.05; Student's t -test. ( B ) RPE1 cells expressing the BCL11A 160–520 peptide were treated with 0, 50 or 100 μM H 2 O 2 and submitted to a clonogenic assay. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Expressing, Single Cell Gel Electrophoresis, Clonogenic Assay

Journal: NAR Cancer

Article Title: The DNA repair function of BCL11A suppresses senescence and promotes continued proliferation of triple-negative breast cancer cells

doi: 10.1093/narcan/zcac028

Figure Lengend Snippet: BCL11A cooperates with RAS to transform primary cells and escape senescence. IMR90 primary fibroblastic cells were infected with retroviruses expressing either HRAS alone or HRAS and BCL11A 160–520 , as indicated. ( A ) Total cell extracts were analyzed by immunoblotting with the indicated antibodies. ( B ) Cells were plated in soft agar 2 days after infection and colonies were counted after 2 weeks Results are an average of three different experiments. Error bars represent standard error. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( C ) IMR90 cells were selected for 5 days with puromycin, cells were then submitted to single cell gel electrophoresis at pH >13. Comet tail moments were scored for at least 100 cells per condition. Error bars represent standard error of three different experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( D ) β-Gal associated senescence was measured 7 days after infection with vectors expressing HRAS or HRAS and BCL11A 160–520 . Values were normalized to the value of empty vector. Error bars represent standard error of three different experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test. ( E ) Expression of the senescence markers P16, P21, IL6 and IL8 was measured by RT-PCR 5 days following infection of IMR90 cells with vectors expressing either HRAS, BCL11A 160–520 , HRAS and BCL11A 160–520 or nothing. Error bars represent standard error of three different experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; Student's t -test.

Article Snippet: 500 μg of nuclear extracts were incubated with either 3 μl of anti-NTHL1 antibody (Proteintech), 3 μl of anti-Pol β antibody (Abcam) or 6 μl of AF3 BCL11A antibody and left to spin overnight at 4°C.

Techniques: Infection, Expressing, Western Blot, Single Cell Gel Electrophoresis, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Food Biochemistry

Article Title: Zang Siwei Qingfei Mixture Alleviates Pulmonary Arterial Hypertension in Rats: Integrated Network Pharmacology and Metabolomics

doi: 10.1155/2024/3435474

Figure Lengend Snippet: Figure 3: Te binding interactions between certain compounds and the binding site of the hub genes protein. (a) Docking models of chrysin in the hub genes protein are depicted in a ribbon format. Ligand-protein interactions were observed between the binding residues of CYP2C19, CASP8, PTK2, VEGFA, FLT4, and TNNI3 (light gray) and chrysin (blue). (b) Ligand–protein interactions involving the binding residues of CYP2C19, CASP8, PTK2, VEGFA, FLT4, TNNI3, and chrysin. Te presence of green dashed lines in the diagram signifes the presence of hydrogen bonds, while the pink dashed lines indicate the occurrence of π-interactions.

Article Snippet: Te primary antibodies used in this study were CASP8 (1: 1000; 13423-1-AP, Proteintech, China), CYP2C19 (1: 1000; 16546-1-AP, Proteintech, China), FLT4 (1: 1000; 20712-1-AP, Proteintech, China), PTK2 (1: 1000; 12636-1-AP, Proteintech, China), TNNI3 (1: 1000; 21652-1-AP, Proteintech, China), and VEGFA (1: 1000; 66828-1-Ig, Proteintech, China).

Techniques: Binding Assay

Journal: Journal of Food Biochemistry

Article Title: Zang Siwei Qingfei Mixture Alleviates Pulmonary Arterial Hypertension in Rats: Integrated Network Pharmacology and Metabolomics

doi: 10.1155/2024/3435474

Figure Lengend Snippet: Figure 4: Efects of administration of ZSQM on the protein expression of hub genes in the lung tissues. (a) Representative western blotting results of CYP2C19, CASP8, PTK2, VEGFA, FLT4, TNNI3, and GAPDH in the lung tissues of the four groups. (b) Ratios of CYP2C19, cleaved-CASP8, PTK2, VEGFA, FLT4, and TNNI3 to GAPDH. Data are presented as mean ± SD (n 5). ∗p < 0.05 vs. the control group. #p < 0.05 vs. the PAH group. ZSQM: Zang Siwei Qingfei mixture; PAH: pulmonary arterial hypertension.

Article Snippet: Te primary antibodies used in this study were CASP8 (1: 1000; 13423-1-AP, Proteintech, China), CYP2C19 (1: 1000; 16546-1-AP, Proteintech, China), FLT4 (1: 1000; 20712-1-AP, Proteintech, China), PTK2 (1: 1000; 12636-1-AP, Proteintech, China), TNNI3 (1: 1000; 21652-1-AP, Proteintech, China), and VEGFA (1: 1000; 66828-1-Ig, Proteintech, China).

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Rab11-FIP1 and Rab11-FIP5 Regulate pIgR/pIgA Transcytosis through TRIM21-Mediated Polyubiquitination

doi: 10.3390/ijms221910466

Figure Lengend Snippet: Rab11−FIP1 colocalizes with pIgA during pIgA transcytosis from the vicinity of centrosome to apical plasma membrane. ( A ) Colocalization of Rab11−FIP1 with the centrosome was detected. Vero−pIgR cells (1 × 10 5 ) were grown on Transwell for 3 days. Cells were fixed with 4% paraformaldehyde and stained with the indicated antibodies before observation by confocal microscopy. Scale bar: 10 µm. ( B , C ) Colocalization of Rab11−FIP1, pIgA with the Golgi apparatus (GM130) or recycling endosomes (Rab11a) was detected. Vero−pIgR cells (1 × 10 5 ) were grown on Transwell for 3 days. An amount of 20 µg pIgA was added or not added to the basal chamber for 10 min at 37 °C and cells were then washed three times. Subsequently, cells were cultivated at 37 °C and harvested at the indicated time points. Finally, cells were fixed with 4% paraformaldehyde and stained with the indicated antibodies before observation by confocal microscopy. Scale bar: 10 µm. ( D , E ) Quantitative analysis of colocalization of Rab11−FIP1, pIgA with GM130 or Rab11a. Statistical analysis was based on colocalization images (covering dozens of cells) using the ImageJ software. ( F ) Analysis of localization of Rab11−FIP1 on the endosomes containing pIgA. Vero−pIgR cells (1 × 10 5 ) were grown on Transwell for 3 days. An amount of 20 µg pIgA was added to the basal chamber for 15 min at 37 °C and cells were then washed three times. Cells were fixed with 4% paraformaldehyde and then were permeabilized by 0.1% Triton X−100 or 20 µg/mL digitonin for 10 min at 4 °C. Finally, cells were stained with the indicated antibodies before observation by confocal microscopy. Scale bar: 10 µm. Data of ( A – F ) are representative of three independent experiments.

Article Snippet: Protease inhibitor cocktail (TargetMol, Shanghai, China); biotin (Thermo Fisher, Waltham, MA, USA); Streptavidin Agarose (YeaSen Biotechnology, Shanghai, China); protein G Sepharose and Glutathione Sepharose (GE Healthcare, Uppsala, Sweden); polybrene (Millipore, Burlington, MA, USA); mouse monoclonal antibodies against Flag (RM1002), HA (RM1004), V5 (RM1006), and β-actin (RM2001) were purchased from Beijing Ray Antibody Biotech (Beijing, China); mouse monoclonal antibodies against pIgR (sc-374343), Rab11-FIP1 (sc-517228), ubiquitin (sc-8017) and Rab11a (sc-166523) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); anti-ubiquitin K11 linkage antibody (MABS107) was purchased from Sigma Aldrich (St. Louis, MO, USA); KDEL (ab12223) and HRP-conjugation Kit (ab102890) were purchased from Abcam (Cambridge, MA, USA); GM130 (610822) was purchased from BD Biosciences (Frankin Lakes, NJ, USA); Rab11-FIP1 (A9215), Rab11-FIP5 (A18142), γ-Tubulin (A9657) and TRIM21 (A1957) were purchased from ABclonal Technology (Wuhan, China); mouse IgA (0106-01), goat anti-mouse IgA (1040-01), goat anti-mouse IgA-AP (1040-04), goat anti-mouse IgA conjugated to Alexa Fluor 647 (1040-31) were purchased from SouthernBiotech (Birmingham, AL, USA); HRP-Streptavidin (405210) was purchased from BioLegend (San Diego, CA, USA).

Techniques: Clinical Proteomics, Membrane, Staining, Confocal Microscopy, Software

Journal: International Journal of Molecular Sciences

Article Title: Rab11-FIP1 and Rab11-FIP5 Regulate pIgR/pIgA Transcytosis through TRIM21-Mediated Polyubiquitination

doi: 10.3390/ijms221910466

Figure Lengend Snippet: A working model on Rab11-FIP1- and Rab11-FIP5-mediated pIgA transcytosis in incompletely polarized cells. In our incompletely polarized cells model, pIgA is recognized and bound by pIgR upon pIgA treatment. Subsequently, the endocytic pIgR–pIgA complex is delivered by Rab11a-positive endosomes. The complex is first transported from the basolateral plasma membrane to the vicinity of the centrosome where Rab11-FIP1 and Rab11-FIP5 bind to it. During the trafficking process, TRIM21 mediates the K11-linked polyubiquitination of Rab11-FIP1 and the K6-linked polyubiquitination of Rab11-FIP5 to promote their activation. With the assistance of Rab11-FIP1 and Rab11-FIP5, pIgR–pIgA complex is further transferred to the apical plasma membrane via Golgi apparatus and then secreted.

Article Snippet: Protease inhibitor cocktail (TargetMol, Shanghai, China); biotin (Thermo Fisher, Waltham, MA, USA); Streptavidin Agarose (YeaSen Biotechnology, Shanghai, China); protein G Sepharose and Glutathione Sepharose (GE Healthcare, Uppsala, Sweden); polybrene (Millipore, Burlington, MA, USA); mouse monoclonal antibodies against Flag (RM1002), HA (RM1004), V5 (RM1006), and β-actin (RM2001) were purchased from Beijing Ray Antibody Biotech (Beijing, China); mouse monoclonal antibodies against pIgR (sc-374343), Rab11-FIP1 (sc-517228), ubiquitin (sc-8017) and Rab11a (sc-166523) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); anti-ubiquitin K11 linkage antibody (MABS107) was purchased from Sigma Aldrich (St. Louis, MO, USA); KDEL (ab12223) and HRP-conjugation Kit (ab102890) were purchased from Abcam (Cambridge, MA, USA); GM130 (610822) was purchased from BD Biosciences (Frankin Lakes, NJ, USA); Rab11-FIP1 (A9215), Rab11-FIP5 (A18142), γ-Tubulin (A9657) and TRIM21 (A1957) were purchased from ABclonal Technology (Wuhan, China); mouse IgA (0106-01), goat anti-mouse IgA (1040-01), goat anti-mouse IgA-AP (1040-04), goat anti-mouse IgA conjugated to Alexa Fluor 647 (1040-31) were purchased from SouthernBiotech (Birmingham, AL, USA); HRP-Streptavidin (405210) was purchased from BioLegend (San Diego, CA, USA).

Techniques: Clinical Proteomics, Membrane, Activation Assay