gapdh band intensity  (Proteintech)

Journal: bioRxiv

Article Title: The SARS-CoV-2 Spike mutation D614G increases entry fitness across a range of ACE2 levels, directly outcompetes the wild type, and is preferentially incorporated into trimers

doi: 10.1101/2020.08.25.267500

Figure Lengend Snippet: A) Percent of HEK-ACE2 cells infected with S D614 - or S G614 -pseudotyped lentivirus, as a function of amount of virus added (ng of p24). Both pseudoviruses expressed GFP, and the percentage of GFP-positive cells was quantified by flow cytometry two days after infection. The infection was done in triplicates. B) The ratio of the percentage of the cells infected by the G614 virus to the percentage of the cells infected by the same amount of the D614 variant. *, **, and *** indicate p-values ≤ .05, .01, and .001, respectively.

Article Snippet: The un-cleaved S expression was slightly lower in the G614 variant than the D614 variant (adjusted band intensity to GAPDH (ABI) 0.95 vs. 1.05, p-value = .009), but the amount of the cleaved S2 fragment was not significantly different between the two variants (ABI 1.09 vs. 0.91, p-value = .36).

Techniques: Infection, Virus, Flow Cytometry, Variant Assay

Journal: bioRxiv

Article Title: The SARS-CoV-2 Spike mutation D614G increases entry fitness across a range of ACE2 levels, directly outcompetes the wild type, and is preferentially incorporated into trimers

doi: 10.1101/2020.08.25.267500

Figure Lengend Snippet: A) Schematic of experimental design. A fixed concentration of the RFP-expressing D614 virus was added to the HEKACE2. At the same time, increasing concentrations of the GFP-expressing D614 or G614 virus was added to the cells to outcompete the RFP-expressing D614 virus. After 1 hour of incubation at 4° C to allow equilibration, the cells were incubated at 37 degrees for 2 days. The percentage of the cells expressing GFP and RFP was quantified using flow cytometry. B) Outcomes of the competition assay. At each ratio of GFP-expressing to RFP-expressing virus added, the percent of total infected cells (RFP + or GFP + ) that were RFP + is reported. Lower percentage of RFP + cells imply that the GFP-expressing virus was more efficient at binding to and replacing the RFP-expressing virus from the surface of the HEKACE2 cells. The competition between GFP-expressing G614 and D614 virus is shown in orange. The control competition of GFP-expressing D614 with RFP-expressing D614 virus is shown in blue. *, **, and *** indicate p-values ≤ .05, .01, and .001, respectively.

Article Snippet: The un-cleaved S expression was slightly lower in the G614 variant than the D614 variant (adjusted band intensity to GAPDH (ABI) 0.95 vs. 1.05, p-value = .009), but the amount of the cleaved S2 fragment was not significantly different between the two variants (ABI 1.09 vs. 0.91, p-value = .36).

Techniques: Concentration Assay, Expressing, Virus, Incubation, Flow Cytometry, Competitive Binding Assay, Infection, Binding Assay, Control

Journal: bioRxiv

Article Title: The SARS-CoV-2 Spike mutation D614G increases entry fitness across a range of ACE2 levels, directly outcompetes the wild type, and is preferentially incorporated into trimers

doi: 10.1101/2020.08.25.267500

Figure Lengend Snippet: An HEK293T cell line expressing ACE2 under the control of a doxycycline-inducible promotor was created and used for infection assays. A) The percentage of the inducible cells expressing ACE2 as a function of the concentration of doxycycline added, quantified using surface staining and flow cytometry. B) The percentage of cells expressing ACE2 in the the parental HEK293T cells and the cell line previously engineered for high ACE2 expression (HEK-ACE2). Cell surface staining and flow cytometry analysis was done similar to panel B and is described in the Methods section. C) The distribution of ACE2 expression levels on the surface of the inducible cells, parental HEK293T cells, and the HEK-ACE2 cells. Cells were stained with an anti-ACE2 antibody followed by a GFP-expressing secondary antibody as in panel B and C. The mean of fluorescent intensity (MFI) in the GFP channel was used to quantify the ACE2 expression level. D) Percent of cells infected with Spike-pseudotyped virus for different Spike variants (G614 vs D614), viral inoculum sizes (7.5 vs 15 ng), and cellular ACE2 expression levels. Infection assays used 30,000 cells and were done in triplicate. Infection was quantified by RFP expression 2 days after virus was added, using flow cytometry. *, **, and *** indicate p-values ≤ .05, .01, and .001, respectively.

Article Snippet: The un-cleaved S expression was slightly lower in the G614 variant than the D614 variant (adjusted band intensity to GAPDH (ABI) 0.95 vs. 1.05, p-value = .009), but the amount of the cleaved S2 fragment was not significantly different between the two variants (ABI 1.09 vs. 0.91, p-value = .36).

Techniques: Expressing, Control, Infection, Concentration Assay, Staining, Flow Cytometry, Virus

Journal: bioRxiv

Article Title: The SARS-CoV-2 Spike mutation D614G increases entry fitness across a range of ACE2 levels, directly outcompetes the wild type, and is preferentially incorporated into trimers

doi: 10.1101/2020.08.25.267500

Figure Lengend Snippet: A) Expression of Spike variants in virus-producing cells. HEK293T cells were transfected with plasmids encoding the D614 and G614 variants of the Spike protein with a fused C-terminal HA tag. Western blot analysis with anti-HA antibody was done to quantify the amount of un-cleaved S and cleaved S2 subunit present in the cell lysate. The transfection and western blot analysis were done in triplicates. B) The amount of the S and S2 subunits was quantified relative to the signal intensity of the GAPDH band. C) The surface expression of each Spike variant on the virus-producing cells. Cells were transfected with plasmids encoding the different Spike variants, D614 and G614 as before. After 2 days, the transfected cells were incubated with a solubilized ACE2 molecule carrying a human IgG1 Fc at the C-terminus, followed by a PE-conjugated mouse-anti-human secondary antibody. The amount of each Spike variant protein on the transfected cells’ surface was quantified as the MFI in the PE channel. The transfection and staining was done in triplicates. D, E) The amount of each Spike variant incorporated into the virion. D614 and G614 viruses were made and concentrated as before. 355 ng p24 equivalent of each virus was lysed and loaded on the gel for western blot analysis using anti-HA antibody. E) The amount of un-cleaved S and cleaved S2 subunit was quantified relative to the Integrase band’s signal intensity. Three independent replicates were analyzed. *, **, and *** indicate p-values ≤ .05, .01, and .001, respectively.

Article Snippet: The un-cleaved S expression was slightly lower in the G614 variant than the D614 variant (adjusted band intensity to GAPDH (ABI) 0.95 vs. 1.05, p-value = .009), but the amount of the cleaved S2 fragment was not significantly different between the two variants (ABI 1.09 vs. 0.91, p-value = .36).

Techniques: Expressing, Virus, Transfection, Western Blot, Variant Assay, Incubation, Staining

Journal: bioRxiv

Article Title: The SARS-CoV-2 Spike mutation D614G increases entry fitness across a range of ACE2 levels, directly outcompetes the wild type, and is preferentially incorporated into trimers

doi: 10.1101/2020.08.25.267500

Figure Lengend Snippet: A, B, C) Differential packaging of Spike monomers into viruses produced from cells containing equal amounts of S D614 and S G614 . Equal molar ratios of plasmids making D614-FLAG and G614-HA or D614-FLAG and G614-HA were transfected into HEK293T cells along with the other necessary constructs to make virions with a mix of Spike proteins. The resulting viruses were collected as before and lysed for western blot analysis. B, C) The western blot analysis for the FLAG-tagged Spike proteins and the HA-tagged Spike proteins was done in triplicates and normalized to the Integrase content. D) Schematic of the protocol used to stabilize and purify the Spike trimers from the surface of the virions. The viruses containing both the FLAG-tagged D614 Spike and either the HA-tagged G614 or the HA-tagged D614 were made from HEK293T cells as described above. The trimers were crosslinked by treating the viral prep with BS3 followed by permeabilization with DDM. The crosslinked Spike trimers were purified by IP using either anti-HA- or anti-FLAG-conjugated magnetic beads. E) Different amounts of each Spike variant are present in chimeric Spike trimers. The vertically-oriented text above the table denotes the virus from which the Spike trimer is purified. The magnetic bead used to purify the Spike trimer is indicated in the first column: F: anti-FLAG-conjugated beads, HA: anti-HA-conjugated beads. The purified trimers were then analyzed by western blot using anti-Flag (top blot) or anti-HA (bottom blot) antibody. F) The relative amount of G614 vs. D614 monomers present in chimeric mutant/wild-type trimers. Anti-Flag-conjugated magnetic beads were used to purify the extracted chimeric Spike trimers. The amount of D614 monomer was quantified (solid blue bar). In addition, the amount of HA-tagged D614 (blue striped bar) and HA-tagged G614 (orange striped bar) was also quantified from Spike trimers extracted from the virions and purified with anti-FLAG beads. The IP and WB analysis were done in triplicates. The Spike protein amounts were normalized to the amount of intra-virion Integrase. D614(HA): viruses containing HA-tagged D614 Spike. D614(FLAG): virus containing the 3xFLAG-tagged Spike. D614 (FLAG)+D614(HA): viruses containing both the 3xFLAG-tagged Spike and the HA-tagged D614 Spike. D614 (FLAG)+G614(HA): viruses containing both the 3xFLAG-tagged D614 Spike and the HA-tagged G614 Spike. *, **, and *** indicate p-values ≤ .05, .01, and .001, respectively.

Article Snippet: The un-cleaved S expression was slightly lower in the G614 variant than the D614 variant (adjusted band intensity to GAPDH (ABI) 0.95 vs. 1.05, p-value = .009), but the amount of the cleaved S2 fragment was not significantly different between the two variants (ABI 1.09 vs. 0.91, p-value = .36).

Techniques: Produced, Transfection, Construct, Western Blot, Purification, Magnetic Beads, Variant Assay, Virus, Mutagenesis