Journal: Biochimica et biophysica acta

Article Title: Inhibition of insulin/PI3K/AKT signaling decreases adipose Sortilin 1 in mice and 3T3-L1 adipocytes

doi: 10.1016/j.bbadis.2017.08.012

Figure Lengend Snippet: A. Confocal microscope of differentiated 3T3-L1 adipocytes stably expressing WT-Sort1-FLAG or S825A-Sort1-FLAG. Cells were cultured in FBS-free medium for 16 h. Sort1-FLAG was stained with anti-FLAG antibody. Blue: DAPI staining of nuclei. B. Confocal microscope of differentiated 3T3-L1 adipocytes stably expressing WT-Sort1-FLAG or S825A-Sort1-FLAG. Cells were cultured in FBS-free medium for 16 h and then treated with 100 nM insulin for 15 min. Sort1 (green) was stained with anti-FLAG antibody. Nuclei were stained with DAPI (Blue). C. Differentiated 3T3-L1 adipocytes were cultured in serum-free medium for 16 h, and treated with 100 nM insulin for 2 min. Surface biotinylation assays were performed. Endogenous biotinylated Sort1 and unbound Sort1 in the flow-through were detected with Sort1 antibody. D, E. Differentiated 3T3-L1 adipocytes expressing WT Sort1-FLAG or S825A Sort1-FLAG were cultured in serum-free medium for 16 h, then treated with 100 nM insulin for 2 and 15 min. Biotinylated Sort1-FLAG and unbound Sort1-FLAG in the flow-through were detected with anti-FLAG antibody. Actin in flow-through was also blotted. F. Differentiated 3T3-L1 adipocytes stably expressing WT-Sort1-FLAG or S825A-Sort1-FLAG were subjected to co-immunoprecipitation with anti-FLAG magnetic beads. Co-precipitated GGA2 and GLUT4 were measured by Western blotting. Differentiated adipocytes without stable Sort1-FLAG expression was subjected to immunoprecipitation as negative controls.

Article Snippet: Antibody against GGA2 (H-175, sc-30103; Lot: K0305) was purchased from Santa Cruz Biotechnology (Dallas, Texas).

Techniques: Microscopy, Stable Transfection, Expressing, Cell Culture, Staining, Immunoprecipitation, Magnetic Beads, Western Blot

Journal: Cell

Article Title: TMEM175 Is an Organelle K(+) Channel Regulating Lysosomal Function.

doi: 10.1016/j.cell.2015.08.002

Figure Lengend Snippet: Figure 3. TMEM175 Is Expressed in Endosomes and Lysosomes (A) Top: hydrophobicity plot of human TMEM175 (hTMEM175). Bottom: a predicted two-repeat six-transmembrane-spanning (2 3 6TM) topology of hTMEM175. Domains that may contain the pore lining are in red. (B) Western blot detecting non-tagged and GFP-tagged hTMEM175 transfected in HEK293T cells. (C and D) Localization of N-terminally YFP-tagged hTMEM175 with RFP-tagged Rab5 (C) or RFP-tagged Lamp1 (D) co-transfected in HeLa cells. (E and F) Co-localization of C-terminally GFP-tagged hTMEM175 with RFP-tagged Rab5 (E) or RFP-tagged Lamp1 (F). Scale bars, 5 mm. See also Figure S2.

Article Snippet: Protein Chemistry and Protein Localization For the western blot used in Figure 3, a polyclonal TMEM175 antibody (Proteintech Group, Cat. 19925-1-AP) was used at 1:500 for western blot analysis.

Techniques: Western Blot, Transfection

Journal: Cell

Article Title: TMEM175 Is an Organelle K(+) Channel Regulating Lysosomal Function.

doi: 10.1016/j.cell.2015.08.002

Figure Lengend Snippet: Figure 6. Bacterial TMEM175 Homologs Form K+-Permeable Channels (A) Hydrophobicity plot (left) and a predicted one-repeat 6TM structure (right) of a bacterial TMEM175 from Chryseobacterium (cbTMEM175). (B–E) Representative whole-cell plasma membrane currents recorded from control (mock-transfected, B and C) HEK293T cells and cells transfected with the cbTMEM175 (D and E) using ramp protocols (B and D; 100 mV to +100 mV in 1 s, Vh = 0 mV) or step protocols (C and E) as illustrated. (F) Averaged current amplitudes at 100 mV with bath cation concentrations indicated. (G) Drug sensitivity presented as the current amplitudes (at 100 mV) after drug application, normalized to the amplitudes before drug applica- tion. Numbers of cells recorded are in parentheses. Data are presented as mean ± SEM. See also Figure S4.

Article Snippet: Protein Chemistry and Protein Localization For the western blot used in Figure 3, a polyclonal TMEM175 antibody (Proteintech Group, Cat. 19925-1-AP) was used at 1:500 for western blot analysis.

Techniques: Clinical Proteomics, Membrane, Control, Transfection

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: NS1 SC09 interacts with LRPPRC. (A) 293T cells were transfected with NS1 SC09 -Flag, NS1 JL89 -Flag, or empty vector. Cell lysates were pulled down using the antibodies against Flag tag. Proteins were then separated using SDS-PAGE and stained with silver, after which they were identified using LC/MS. (B) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to HA-IP. (C) HeLa cells were co-transfected with LRPPRC-Flag and NS1 SC09 -HA, NS1 JL89 -HA, or NS1 WSN -HA. Twenty-four h later, cells were assessed for the localization of NS1, LRPPRC, and TOMM20. Cyan color indicated the colocalization of LRPPRC and TOMM20 and white color indicated the colocalization of LRPPRC, NS1, and TOMM20. Scale bar: 5 μm. It was representative of 20 cells. (D) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Transfection, Plasmid Preparation, FLAG-tag, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: Summary annotated proteins identified to interact with NS1 SC09 -flag but not NS1JL89-flag and Vec-flag.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Virus, Binding Assay

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: The key domain responsible for the interaction between NS1 SC09 and LRPPRC. (A) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (B) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to HA-IP. (C) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Transfection

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: LRPPRC is a key factor that regulates the replication of IAVs through the autophagy pathway and is antagonized by NS1 SC09 . (A and B) 293T cells, 293T_LRPPRC cells, and LPRRPC KO cells were infected with WSN at the MOI of 0.01. The supernatants were sampled at 0, 12, 24, and 48 h post-infection, and the virus titers were determined using endpoint titration in MDCK cells. Cell lysates at 0, 12, 24, and 48 h post-infection were analyzed using western blotting. (C and D) 293T cells, 293T_LRPPRC cells, and LPRRPC KO cells were infected with WSN at the MOI of 5.0, in the presence or absence of rapamycin (20 μm) or 3-MA (5 mM). The supernatants were sampled at 24 h post-infection, and the virus titers were determined using endpoint titration in MDCK cells. The cell lysates were analyzed using western blotting. (E) 293T cells, 293T_LRPPRC cells, and LPRRPC KO cells were transfected with mRFP-GFP-LC3B and then infected with WSN at the MOI of 0.1. Cells were analyzed to assess the formation of autophagosomes. Fluorescence signals indicated the expression of RFP and GFP (yellow color: incomplete autophagy, red color: complete autophagy). Scale bar: 10 μm. It was representative of 20 cells. (F) The graph shows the quantification of mRFP+GFP+ autophagosomes and mRFP+GFP- autolysosomes by taking the average number of dots in 20 cells (n=average number of dots in 20 cells). (G and H) 293T cells and LPRRPC KO cells were infected with WSN-NS1 SC09 or WSN at the MOI of 0.1. The supernatants were sampled at 24 h post-infection, and the virus titers were determined using endpoint titration in MDCK cells. Cell lysates were analyzed using western blotting. (I) 293T cells and LPRRPC KO cells were transfected with NS1 SC09 -HA or NS1 WSN -HA. 24 h post-transfection, cell lysates were analyzed using western blotting. Error bars indicate the SD from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Infection, Virus, Titration, Western Blot, Transfection, Fluorescence, Expressing

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: The interaction between NS1 SC09 and LRPPRC results in initiation of BECN1-dependent autophagy. (A) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (B) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (C) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (D) 293T cells were transfected with empty vector, NS1 SC09 -HA, NS1 JL89 -HA, or NS1 WSN -HA. Cell lysates were prepared and immunoprecipitated with the anti-BECN1 antibody or control IgG. (E) The indicated plasmids above were co-transfected into 293T cells. Cell lysates were subjected to Flag-IP. (F) 293T cells were transfected with empty vector, NS1 SC09 -HA, NS1 JL89 -HA, or NS1 WSN -HA. Cell lysates were prepared and immunoprecipitated with the anti-PIK3C3 antibody or control IgG. (G) The indicated plasmids above were co-transfected into 293T cells. Cell lysates were subjected to Flag-IP.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Control

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: A proposed model for the pathway of autophagy induced by NS1 pdm09 . In the normal physiological state, LRPPRC interacts with the BECN1-BCL2 heterodimer, forming a stable complex in the mitochondria, thereby inhibiting BECN1-dependent autophagy. During A/(H1N1) pdm09 infection, the NS1 pdm09 promotes viral replication by enhancing autophagy which is mainly attributed to the inhibitory effect of NS1 pdm09 on the negative regulation of autophagy by LRPPRC. The interaction between NS1 pdm09 and LRPPRC competitively blocks the interaction of LRPPRC with BECN1-BCL2 heterodimer, resulting in degradation of BCL2 and increased recruitment of BECN1 by PIK3C3, and then induction of the initiation of autophagy.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Infection

Journal: Cell stem cell

Article Title: Formation of Human Neuroblastoma in Mouse-Human Neural Crest Chimeras

doi: 10.1016/j.stem.2020.02.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For immunostainings, samples were blocked with 2% BSA and incubated with primary antibodies including Rabbit anti-ALK (1:200, Cell Signaling technology (CST), 3633), Rabbit anti-MYCN (1:50 84406; 1:100 51705, CST), Rabbit anti-CD3 (1:300, RM9107S0, Thermo Fisher), Rabbit anti-CD8a (1:300, 98941, CST), Rabbit anti-FoxP3 (1:75, MAB8214, R&D), Mouse anti-Il2ra (CD25, 1:100, NB600–564, Novus), Rabbit anti mouse Tim-3 (1:200, 83882, CST), Rabbit anti-human-PD-L1 (1:300, 13684, CST), Sheep anti-human-CD47 (1:100, AF4670SP, R&D), Mouse anti- IFNγ (1:200, DB-1, BioLegend), Rat anti-mouse F4/80 (1:100, MF48000, Thermo Fisher), Rabbit anti-γH2AX (1:300, ab2893, Abcam), Rabbit anti-human-Ki67 (1:20, PA516785, Thermo Fisher), Rabbit anti-Chromogranin A (1:500, NB120–15160, Novus), Rabbit anti-Synaptophysin (1:200, 36406, CST), Mouse anti-human-Nestin (1:300, ab176571, Abcam), Rabbit anti-TH (1:500, {"type":"entrez-protein","attrs":{"text":"P40101","term_id":"731386","term_text":"P40101"}} P40101 –150, Pel-Freez), Rabbit anti-pERK1/2 (1:400, 4370, SCT), Rabbit anti-p-p70 S6 (1:300, 9205, CST), Rabbit anti-Peripherin (1:500, ab4666, Abcam), Rabbit anti-human neurofilament (1:100, 160kD, ab92539, Abcam), anti-eGFP (1:1000, GFP-1020 Aves Labs; 1:300 AF647-Alpaca nanobody; 1:5000, ab290, Abcam), mouse anti-HNK-1 (1:50, C6680, MilliporeSigma) and rabbit anti-p75 (1:175, G3231, Promega) overnight at 4°C followed by appropriate secondary antibody incubation for 1–2 h (for IF: A31571, A21235, A21202, A11001, A21206, A11008, {"type":"entrez-protein","attrs":{"text":"A31573","term_id":"87384","term_text":"pir||A31573"}} A31573 , A21244, A11015 or A21209, Thermo Fisher; 703–545-155, 115–585-044, Jax ILR; for IHC: Rabbit-on-Rodent and Mouse-on-Mouse HRP-Polymer, RMR622; MM620, Biocare, or Biotinylated anti-rabbit IgG, BA-1000 Vector).

Techniques: Plasmid Preparation, Microarray, Recombinant, Isolation, SYBR Green Assay, Sequencing, Software