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A) Ser5 and mSer5 amino acid sequences containing the conserved lysine (K130, K131) are aligned. The ICL1 and TMD3 regions are indicated. Dots indicate identical residues. B) Ser5 and mSer5 WT proteins and their lysine mutants (K130R, K131R) with a GFP-tag were expressed in HeLa cells and detected by confocal microscopy (scale bar 5 µm). C) Ser5 and mSer5 proteins and their lysine mutants were expressed in HEK293T cells and their expression in Mem and Cyto fractions were analyzed by WB. D) MLVΔglycoGag and HIV-1ΔNef luciferase-reporter viruses were produced from HEK293T cells in the presence of indicated Ser5 proteins. After infection of NIH3T3 or TZM-bI cells, their infectivity was compared by measuring intracellular firefly luciferase activity. Results are normalized by the p30 Gag or p24 Gag protein levels of virions and presented as relative values, with the infectivity of viruses produced in a control (Ctrl) vector set as 100. Error bars indicate SEMs calculated from two experiments. E) Ser5, mSer5, and their lysine mutants were expressed with Nef or glycoMA in HEK293T cells and protein expression was analyzed by WB. 19K/R, Ser5 with all 19 lysine residues replaced with arginine; 23K/R, mSer5 with all 23 lysine residues replaced with arginine. F) Ser5 and K130R were expressed with Nef or glycoMA in HEK293T cells, where Cas9 and KLHL20 - or Cul3 -specific sgRNA was also expressed. Protein expression was detected by WB. G) Ser5-GFP (wild-type, WT) and its mutants (S360A, S249A) were expressed with HA-tagged Nef or glycoMA in HeLa cells. Cells were stained with DAPI (4′,6-diamidino-2-phenylindole) for the nuclei (blue) and with anti-HA followed by Alexa Fluor 594-conjugated goat anti-mouse antibodies for viral proteins and observed by confocal microscopy (scale bar 5 µm). H) Ser5 and the two mutants were expressed with Nef or glycoMA in HEK293T cells and protein expression was analyzed by WB. I) Ser5 was expressed with a CCNK -shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells and protein expression was analyzed by WB. J) Ser5 was expressed with a <t>CDK13</t> -shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells and protein expression was analyzed by WB. All experiments were at least repeated twice, and similar results were obtained.
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A) Ser5 and mSer5 amino acid sequences containing the conserved lysine (K130, K131) are aligned. The ICL1 and TMD3 regions are indicated. Dots indicate identical residues. B) Ser5 and mSer5 WT proteins and their lysine mutants (K130R, K131R) with a GFP-tag were expressed in HeLa cells and detected by confocal microscopy (scale bar 5 µm). C) Ser5 and mSer5 proteins and their lysine mutants were expressed in HEK293T cells and their expression in Mem and Cyto fractions were analyzed by WB. D) MLVΔglycoGag and HIV-1ΔNef luciferase-reporter viruses were produced from HEK293T cells in the presence of indicated Ser5 proteins. After infection of NIH3T3 or TZM-bI cells, their infectivity was compared by measuring intracellular firefly luciferase activity. Results are normalized by the p30 Gag or p24 Gag protein levels of virions and presented as relative values, with the infectivity of viruses produced in a control (Ctrl) vector set as 100. Error bars indicate SEMs calculated from two experiments. E) Ser5, mSer5, and their lysine mutants were expressed with Nef or glycoMA in HEK293T cells and protein expression was analyzed by WB. 19K/R, Ser5 with all 19 lysine residues replaced with arginine; 23K/R, mSer5 with all 23 lysine residues replaced with arginine. F) Ser5 and K130R were expressed with Nef or glycoMA in HEK293T cells, where Cas9 and KLHL20 - or Cul3 -specific sgRNA was also expressed. Protein expression was detected by WB. G) Ser5-GFP (wild-type, WT) and its mutants (S360A, S249A) were expressed with HA-tagged Nef or glycoMA in HeLa cells. Cells were stained with DAPI (4′,6-diamidino-2-phenylindole) for the nuclei (blue) and with anti-HA followed by Alexa Fluor 594-conjugated goat anti-mouse antibodies for viral proteins and observed by confocal microscopy (scale bar 5 µm). H) Ser5 and the two mutants were expressed with Nef or glycoMA in HEK293T cells and protein expression was analyzed by WB. I) Ser5 was expressed with a CCNK -shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells and protein expression was analyzed by WB. J) Ser5 was expressed with a <t>CDK13</t> -shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells and protein expression was analyzed by WB. All experiments were at least repeated twice, and similar results were obtained.
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A) Ser5 and mSer5 amino acid sequences containing the conserved lysine (K130, K131) are aligned. The ICL1 and TMD3 regions are indicated. Dots indicate identical residues. B) Ser5 and mSer5 WT proteins and their lysine mutants (K130R, K131R) with a GFP-tag were expressed in HeLa cells and detected by confocal microscopy (scale bar 5 µm). C) Ser5 and mSer5 proteins and their lysine mutants were expressed in HEK293T cells and their expression in Mem and Cyto fractions were analyzed by WB. D) MLVΔglycoGag and HIV-1ΔNef luciferase-reporter viruses were produced from HEK293T cells in the presence of indicated Ser5 proteins. After infection of NIH3T3 or TZM-bI cells, their infectivity was compared by measuring intracellular firefly luciferase activity. Results are normalized by the p30 Gag or p24 Gag protein levels of virions and presented as relative values, with the infectivity of viruses produced in a control (Ctrl) vector set as 100. Error bars indicate SEMs calculated from two experiments. E) Ser5, mSer5, and their lysine mutants were expressed with Nef or glycoMA in HEK293T cells and protein expression was analyzed by WB. 19K/R, Ser5 with all 19 lysine residues replaced with arginine; 23K/R, mSer5 with all 23 lysine residues replaced with arginine. F) Ser5 and K130R were expressed with Nef or glycoMA in HEK293T cells, where Cas9 and KLHL20 - or Cul3 -specific sgRNA was also expressed. Protein expression was detected by WB. G) Ser5-GFP (wild-type, WT) and its mutants (S360A, S249A) were expressed with HA-tagged Nef or glycoMA in HeLa cells. Cells were stained with DAPI (4′,6-diamidino-2-phenylindole) for the nuclei (blue) and with anti-HA followed by Alexa Fluor 594-conjugated goat anti-mouse antibodies for viral proteins and observed by confocal microscopy (scale bar 5 µm). H) Ser5 and the two mutants were expressed with Nef or glycoMA in HEK293T cells and protein expression was analyzed by WB. I) Ser5 was expressed with a CCNK -shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells and protein expression was analyzed by WB. J) Ser5 was expressed with a <t>CDK13</t> -shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells and protein expression was analyzed by WB. All experiments were at least repeated twice, and similar results were obtained.
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A) Ser5 and mSer5 amino acid sequences containing the conserved lysine (K130, K131) are aligned. The ICL1 and TMD3 regions are indicated. Dots indicate identical residues. B) Ser5 and mSer5 WT proteins and their lysine mutants (K130R, K131R) with a GFP-tag were expressed in HeLa cells and detected by confocal microscopy (scale bar 5 µm). C) Ser5 and mSer5 proteins and their lysine mutants were expressed in HEK293T cells and their expression in Mem and Cyto fractions were analyzed by WB. D) MLVΔglycoGag and HIV-1ΔNef luciferase-reporter viruses were produced from HEK293T cells in the presence of indicated Ser5 proteins. After infection of NIH3T3 or TZM-bI cells, their infectivity was compared by measuring intracellular firefly luciferase activity. Results are normalized by the p30 Gag or p24 Gag protein levels of virions and presented as relative values, with the infectivity of viruses produced in a control (Ctrl) vector set as 100. Error bars indicate SEMs calculated from two experiments. E) Ser5, mSer5, and their lysine mutants were expressed with Nef or glycoMA in HEK293T cells and protein expression was analyzed by WB. 19K/R, Ser5 with all 19 lysine residues replaced with arginine; 23K/R, mSer5 with all 23 lysine residues replaced with arginine. F) Ser5 and K130R were expressed with Nef or glycoMA in HEK293T cells, where Cas9 and KLHL20 - or Cul3 -specific sgRNA was also expressed. Protein expression was detected by WB. G) Ser5-GFP (wild-type, WT) and its mutants (S360A, S249A) were expressed with HA-tagged Nef or glycoMA in HeLa cells. Cells were stained with DAPI (4′,6-diamidino-2-phenylindole) for the nuclei (blue) and with anti-HA followed by Alexa Fluor 594-conjugated goat anti-mouse antibodies for viral proteins and observed by confocal microscopy (scale bar 5 µm). H) Ser5 and the two mutants were expressed with Nef or glycoMA in HEK293T cells and protein expression was analyzed by WB. I) Ser5 was expressed with a CCNK -shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells and protein expression was analyzed by WB. J) Ser5 was expressed with a <t>CDK13</t> -shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells and protein expression was analyzed by WB. All experiments were at least repeated twice, and similar results were obtained.
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Image Search Results


A) Ser5 and mSer5 amino acid sequences containing the conserved lysine (K130, K131) are aligned. The ICL1 and TMD3 regions are indicated. Dots indicate identical residues. B) Ser5 and mSer5 WT proteins and their lysine mutants (K130R, K131R) with a GFP-tag were expressed in HeLa cells and detected by confocal microscopy (scale bar 5 µm). C) Ser5 and mSer5 proteins and their lysine mutants were expressed in HEK293T cells and their expression in Mem and Cyto fractions were analyzed by WB. D) MLVΔglycoGag and HIV-1ΔNef luciferase-reporter viruses were produced from HEK293T cells in the presence of indicated Ser5 proteins. After infection of NIH3T3 or TZM-bI cells, their infectivity was compared by measuring intracellular firefly luciferase activity. Results are normalized by the p30 Gag or p24 Gag protein levels of virions and presented as relative values, with the infectivity of viruses produced in a control (Ctrl) vector set as 100. Error bars indicate SEMs calculated from two experiments. E) Ser5, mSer5, and their lysine mutants were expressed with Nef or glycoMA in HEK293T cells and protein expression was analyzed by WB. 19K/R, Ser5 with all 19 lysine residues replaced with arginine; 23K/R, mSer5 with all 23 lysine residues replaced with arginine. F) Ser5 and K130R were expressed with Nef or glycoMA in HEK293T cells, where Cas9 and KLHL20 - or Cul3 -specific sgRNA was also expressed. Protein expression was detected by WB. G) Ser5-GFP (wild-type, WT) and its mutants (S360A, S249A) were expressed with HA-tagged Nef or glycoMA in HeLa cells. Cells were stained with DAPI (4′,6-diamidino-2-phenylindole) for the nuclei (blue) and with anti-HA followed by Alexa Fluor 594-conjugated goat anti-mouse antibodies for viral proteins and observed by confocal microscopy (scale bar 5 µm). H) Ser5 and the two mutants were expressed with Nef or glycoMA in HEK293T cells and protein expression was analyzed by WB. I) Ser5 was expressed with a CCNK -shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells and protein expression was analyzed by WB. J) Ser5 was expressed with a CDK13 -shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells and protein expression was analyzed by WB. All experiments were at least repeated twice, and similar results were obtained.

Journal: bioRxiv

Article Title: Murine Leukemia Virus GlycoGag Antagonizes SERINC5 via ER-phagy Receptor RETREG1

doi: 10.1101/2025.03.06.641798

Figure Lengend Snippet: A) Ser5 and mSer5 amino acid sequences containing the conserved lysine (K130, K131) are aligned. The ICL1 and TMD3 regions are indicated. Dots indicate identical residues. B) Ser5 and mSer5 WT proteins and their lysine mutants (K130R, K131R) with a GFP-tag were expressed in HeLa cells and detected by confocal microscopy (scale bar 5 µm). C) Ser5 and mSer5 proteins and their lysine mutants were expressed in HEK293T cells and their expression in Mem and Cyto fractions were analyzed by WB. D) MLVΔglycoGag and HIV-1ΔNef luciferase-reporter viruses were produced from HEK293T cells in the presence of indicated Ser5 proteins. After infection of NIH3T3 or TZM-bI cells, their infectivity was compared by measuring intracellular firefly luciferase activity. Results are normalized by the p30 Gag or p24 Gag protein levels of virions and presented as relative values, with the infectivity of viruses produced in a control (Ctrl) vector set as 100. Error bars indicate SEMs calculated from two experiments. E) Ser5, mSer5, and their lysine mutants were expressed with Nef or glycoMA in HEK293T cells and protein expression was analyzed by WB. 19K/R, Ser5 with all 19 lysine residues replaced with arginine; 23K/R, mSer5 with all 23 lysine residues replaced with arginine. F) Ser5 and K130R were expressed with Nef or glycoMA in HEK293T cells, where Cas9 and KLHL20 - or Cul3 -specific sgRNA was also expressed. Protein expression was detected by WB. G) Ser5-GFP (wild-type, WT) and its mutants (S360A, S249A) were expressed with HA-tagged Nef or glycoMA in HeLa cells. Cells were stained with DAPI (4′,6-diamidino-2-phenylindole) for the nuclei (blue) and with anti-HA followed by Alexa Fluor 594-conjugated goat anti-mouse antibodies for viral proteins and observed by confocal microscopy (scale bar 5 µm). H) Ser5 and the two mutants were expressed with Nef or glycoMA in HEK293T cells and protein expression was analyzed by WB. I) Ser5 was expressed with a CCNK -shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells and protein expression was analyzed by WB. J) Ser5 was expressed with a CDK13 -shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells and protein expression was analyzed by WB. All experiments were at least repeated twice, and similar results were obtained.

Article Snippet: Rabbit anti-CycK (Ab85854) was purchased from Abcam; rabbit anti-CDC2L5 (NB100-68268) and KLHL20 (NBP1-79570-20) were from Novus; rabbit anti-LAMP2a (AF1036) was from Beyotime; Alexa Fluor 594-conjugated goat anti-mouse (A11032) and Alexa Fluor 488-conjugated goat anti-mouse (A11029) were from Invitrogen.

Techniques: Confocal Microscopy, Expressing, Luciferase, Produced, Infection, Activity Assay, Control, Plasmid Preparation, Staining, shRNA