bend 3 atcc cat  (ATCC)

Journal: Cell reports

Article Title: Brain endothelial PTEN/AKT/NEDD4-2/MFSD2A axis regulates blood-brain barrier permeability.

doi: 10.1016/j.celrep.2021.109327

Figure Lengend Snippet: Figure 4. PTEN/AKT regulates MFSD2A protein expression via NEDD4-2 (A) Western blot analyses of the MFSD2A, p-AKT1, AKT1, p-NEDD4-2 (S342), p-NEDD4-2 (S448), and GAPDH levels in MFSD2A-expressing HEK293T cells treated with or without IGF-1. MG132 was added 8 h before collection. IGF-1 treatment increased p-AKT1 level and decreased MFSD2A level. (B) Immunostaining analyses for EGFP (green) and Myc (red) in HEK293T cells transfected with the MFSD2A-EGFP and NEDD4-2-Myc plasmids. Scale bar, 20 mm. (C) Western blot analyses of the MFSD2A, p-AKT1, AKT1, p-NEDD4-2(S342/S448), NEDD4-2, and GAPDH levels in MFSD2A-expressing HEK293T cells treated with IGF-1 or LY294002. IGF-1 treatment increased the protein levels of p-AKT1 and p-NEDD4-2(S342/S448) and decreased MFSD2A levels in a time-dependent manner. LY294002 treatment decreased p-AKT1 and p-NEDD4-2(S342/S448) levels and increased MFSD2A levels in a dose-dependent manner. (D) Western blot analyses of the MFSD2A, p-AKT1, AKT1, p-NEDD4-2(S342/S448), NEDD4-2, and GAPDH levels in MFSD2A-expressing HEK293T cells treated with IGF-1 and siRNAs targeting NEDD4-2. siNEDD4-2 treatment increased the MFSD2A levels and blocked the decrease in MFSD2A protein induced by IGF-1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER TSA plus Fluorescence Kit PerkinElmer Cat#NEL741001KT Experimental models: Cell lines HEK293T cells ATCC Cat#CRL-11268; RRID: CVCL_1926 BEnd.3 cells ATCC Cat#CRL-2299, RRID:CVCL_0170 Experimental models: Organisms/strains Mouse: Ptenfl/fl Suzuki et al., 2001 N/A Mouse: Akt1fl/fl Di et al., 2012 N/A Mouse: SP-A-Cre Li et al., 2011 N/A Mouse: Cdh5-CreERT2 Wang et al., 2010 RRID:IMSR_TAC:13073 Mouse: ROSA26EYFP Jackson Laboratory RRID:IMSR_JAX:006148 Oligonucleotides Pten floxed mouse genotyping primers: Fwd: 50-CTCCTCTACTCCATTCTTCCC-30 This paper N/A Pten floxed mouse genotyping primers: Rev:50 -ACTCCCACCAATGAACAAAC-30 This paper N/A Akt1 floxed mouse genotyping primers: Fwd: 50-GGGATCAGCAGTTGAAGGACAGA-30 This paper N/A Akt1 floxed mouse genotyping primers: Rev: 50 -GCCAGGAATACAGCATGAGCCAC-30 This paper N/A SP-A-Cre mouse genotyping primers: Fwd: 50-GCATCGACCGGTAATGCAGGC-30 This paper N/A SP-A-Cre mouse genotyping primers: Rev: 50 -TTTTCTGAATGGCAGTGCATCTT-30 This paper N/A Cdh5-CreERT2 mouse genotyping primers: Fwd: 50-GCCTGCATTACCGGTCGATGC-30 This paper N/A Cdh5-CreERT2 mouse genotyping primers: Rev: 50-CAGGGTGTTATAAGCAATCCC-30 This paper N/A Caveolin1 shRNA: 50-TGAAGCTATTGGCAAGATATT-30 This paper N/A Recombinant DNA AAV-BR1-CAG-EGFP Körbelin et al., 2016 N/A AAV-BR1-CMV-EGFP-ShCav-1 This paper N/A AAV-BR1-CMV-tdTomato-MFSD2A-HA This paper N/A pLenti-MFSD2A-HA This paper N/A Software and algorithms LSM 880 Confocal Zeiss N/A GraphPad Prism 7 GraphPad Software RRID: SCR_002798; https://www.graphpad.com/ ImageJ National Institute of Health RRID: SCR_003070; https://imagej.net ZEN Zeiss https://www.zeiss.com/microscopy/us/products/ microscope-software/zen.html

Techniques: Expressing, Western Blot, Immunostaining, Transfection

Journal: Cell reports

Article Title: Brain endothelial PTEN/AKT/NEDD4-2/MFSD2A axis regulates blood-brain barrier permeability.

doi: 10.1016/j.celrep.2021.109327

Figure Lengend Snippet: Figure 5. NEDD4-2 interacts with MFSD2A and regulates its stability by polyubiquitination (A and B) MFSD2A-expressing HEK293T cells were treated with cycloheximide (CHX, 10 mg/mL) and collected at the indicated times for western blot (A). Quantification of the MFSD2A levels relative to GAPDH is shown (B). Data are means ± SEM (n = 4 per group, **p < 0.01). (C and D) HEK293T cells were transfected with a constant amount of HA-MFSD2A and increasing amounts of Myc-NEDD4-2 (C). Cells were treated with MG132 8 h before collection. Quantification of MFSD2A levels relative to GAPDH is shown (D). Data are means ± SEM (n = 4 per group, **p < 0.01, *p < 0.05). (E) HEK293T cells were transfected with plasmids encoding HA-MFSD2A and Myc-NEDD4-2 and then coimmunoprecipitated with a HA (left panel) or MYC (right panel) antibody. NEDD4-2 interactions with MFSD2A were analyzed by western blot. (F) HEK293T cells were transfected with plasmids encoding HA-MFSD2A, His-Ubiquitin, and increasing amounts (0 mg, 0.5 mg, 1.5 mg, and 3.0 mg) of Myc-NEDD4- 2 were coimmunoprecipitated with the HA antibody. MFSD2A ubiquitin was analyzed by western blotting.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER TSA plus Fluorescence Kit PerkinElmer Cat#NEL741001KT Experimental models: Cell lines HEK293T cells ATCC Cat#CRL-11268; RRID: CVCL_1926 BEnd.3 cells ATCC Cat#CRL-2299, RRID:CVCL_0170 Experimental models: Organisms/strains Mouse: Ptenfl/fl Suzuki et al., 2001 N/A Mouse: Akt1fl/fl Di et al., 2012 N/A Mouse: SP-A-Cre Li et al., 2011 N/A Mouse: Cdh5-CreERT2 Wang et al., 2010 RRID:IMSR_TAC:13073 Mouse: ROSA26EYFP Jackson Laboratory RRID:IMSR_JAX:006148 Oligonucleotides Pten floxed mouse genotyping primers: Fwd: 50-CTCCTCTACTCCATTCTTCCC-30 This paper N/A Pten floxed mouse genotyping primers: Rev:50 -ACTCCCACCAATGAACAAAC-30 This paper N/A Akt1 floxed mouse genotyping primers: Fwd: 50-GGGATCAGCAGTTGAAGGACAGA-30 This paper N/A Akt1 floxed mouse genotyping primers: Rev: 50 -GCCAGGAATACAGCATGAGCCAC-30 This paper N/A SP-A-Cre mouse genotyping primers: Fwd: 50-GCATCGACCGGTAATGCAGGC-30 This paper N/A SP-A-Cre mouse genotyping primers: Rev: 50 -TTTTCTGAATGGCAGTGCATCTT-30 This paper N/A Cdh5-CreERT2 mouse genotyping primers: Fwd: 50-GCCTGCATTACCGGTCGATGC-30 This paper N/A Cdh5-CreERT2 mouse genotyping primers: Rev: 50-CAGGGTGTTATAAGCAATCCC-30 This paper N/A Caveolin1 shRNA: 50-TGAAGCTATTGGCAAGATATT-30 This paper N/A Recombinant DNA AAV-BR1-CAG-EGFP Körbelin et al., 2016 N/A AAV-BR1-CMV-EGFP-ShCav-1 This paper N/A AAV-BR1-CMV-tdTomato-MFSD2A-HA This paper N/A pLenti-MFSD2A-HA This paper N/A Software and algorithms LSM 880 Confocal Zeiss N/A GraphPad Prism 7 GraphPad Software RRID: SCR_002798; https://www.graphpad.com/ ImageJ National Institute of Health RRID: SCR_003070; https://imagej.net ZEN Zeiss https://www.zeiss.com/microscopy/us/products/ microscope-software/zen.html

Techniques: Expressing, Western Blot, Transfection, Ubiquitin Proteomics