Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A-B) Confocal images of YFP-PrP* NRK cells at steady state. Scale bar represents 10 µm. (A) Immunofluorescence image of endogenous calnexin (CNX) in a YFP-PrP* NRK cell. The nucleus was stained with Hoechst. (B) Immunofluorescence image of cell-surface YFP-PrP* using anti-GFP antibody on cells that were not permeabilized. The nucleus was stained with Hoechst. (C-D) Confocal images of GFP-CD59 (C94S) NRK cells at steady state. Scale bar represents 10 µm. (C) Immunofluorescence image against endogenous calnexin (CNX) in GFP-CD59 (C94S). The nucleus was stained with Hoechst. (D) Immunofluorescence of cell-surface GFP-CD59 (C94S) using anti-GFP antibody on cells that were not permeabilized. The nucleus was stained with Hoechst. (E) Western blots of GFP-tag co-immunoprecipitates from the parental untransfected NRK cells (P) or stably transfected YFP-PrP* NRK cells at steady state. Blots were probed for GFP for YFP-PrP*, and probed for endogenous calnexin (CNX), TMP21, TMED2 and TMED9. (F) Western blots of GFP-tag co-immunoprecipitates from parental untransfected NRK cells (P) or stably transfected GFP-CD59 (C94S) NRK cells (n=1). Blots were probed for GFP for GFP-CD59 (C94S), and probed for endogenous calnexin (CNX), TMP21, TMED2 and TMED9.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Immunofluorescence, Staining, Western Blot, Stable Transfection, Transfection

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Time-lapse imaging sequence collected immediately after thapsigargin (TG)-treatment of a typical YFP-PrP* NRK cell that was co-expressing ER-marker, Cerulean-calnexin (CER-CNX). Scale bar represents 10 µm. (B) Plot of the average Pearson’s r values between YFP-PrP* and CER-CNX for 9 individual cells across the 0 and 20 min time points of time-lapses collected after addition of TG, as shown in (A). Pearson’s colocalization coefficients, r, were measured between YFP-PrP* and CER-CNX within the boundaries of the cell. For each image, the boundary of the cell was revealed by temporarily increasing the gain for CER-CNX. The Pearson’s r value for individual cells are color-coded. (C) Time-lapse imaging sequence collected immediately after TG-treatment of a typical YFP-PrP* NRK cell that was co-expressing the Golgi marker, CER-GalT. Scale bar represents 10 µm. (D) Plot of the average Pearson’s r values between YFP-PrP* and CER-GalT for 10 individual cells across the T0 and T20min time points of time-lapses collected after addition of TG, as shown in (C). Pearson’s colocalization coefficients, r, were measured between YFP-PrP* and CER-GalT within the boundaries of the cell. For each image, the boundary of the cell was revealed by temporarily maximizing the gain for YFP-PrP*. The Pearson’s r value for individual cells are color-coded. (E) Time-lapse images collected immediately after TG-treatment of a typical YFP-PrP* NRK cell that was co-expressing CER-TMED9 and FusionRed (FusRed)-SiT as a Golgi-marker. This figure panel is also provided as Video 1. Scale bar represents 10 µm. (F) Plot of the average Pearson’s r values between YFP-PrP* and FusRed-SiT or CER-TMED9 and FusRed-SiT, as indicated, for 6 individual cells across the 0 and 20min time points of time-lapses collected after addition of TG, as shown in (E). Pearson’s colocalization coefficients, r, were measured between YFP-PrP* and FusRed-SiT or CER-TMED9 and FusRed-SiT, within the boundaries of the cell. For each data point at 0 and 20 min time points, the boundary of the cell was revealed by temporarily maximizing the gain for YFP-PrP*. The Pearson’s r value for individual cells are color-coded.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Imaging, Sequencing, Expressing, Marker

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Representative western blots of GFP-tag co-immunoprecipitations from the parental untransfected NRK cells (P) or stably transfected YFP-PrP* NRK cells (n=3 biological replicates). YFP-PrP* NRK cells were treated with thapsigargin (TG) and collected for co-immunoprecipitation at the indicated time points. In addition to using anti-GFP antibody to detect co-immunoprecipitation of YFP-PrP*, blots were probed for calnexin (CNX), TMED9 and TMP21. “L” indicates the lane that the ladder was loaded in. (B) Bar graph representing the mean band intensity for CNX, TMED9 and TMP21 in the eluates from 3 independently performed experiments as shown in (A). For each time point, CNX, TMED9 and TMP21 eluate band intensities were double normalized. First, they were normalized by the band intensity of eluate YFP-PrP*. Second, they were normalized against eluate time 0 band intensity of each protein probed. Error bars represent standard deviations of the mean of 3 independent experiments. Symbols were coded for each experiment. Statistics were calculated from unpaired t-test with Welch’s correction with * indicated p<0.05 and ** indicated p<0.01.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Western Blot, Stable Transfection, Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Representative western blots of control (siTMED9 –) or siTMED9-treated (siTMED9 +) YFP-PrP* NRK cells that were either untreated (TG –) or treated with TG for 90 min (TG +) (n=3 biological replicates). Blots were probed for TMED9, PrP to detect YFP-PrP* and GAPDH. Each lane was numbered to facilitate cross-referencing with the quantification shown in (B). (B) Bar graph representing the mean band intensity for YFP-PrP* from 3 biological replicates as shown in (A). For each condition, YFP-PrP* band intensities were double normalized. First, they were normalized by the band intensity of GAPDH. Second, they were normalized against the untreated control (“siTMED9 – TG – “) band intensity. Error bars represent standard deviation of the mean. Symbols were coded for each independently performed experiment. Statistics were calculated from unpaired t-test with Welch’s correction with * indicated p<0.05 and ** indicated p<0.01. The bars were numbered 1-4 to facilitate cross-referencing with the representative blot in (A). (C) Time-lapse images of control (CTL) or TMED9 siRNA (siTMED9)-treated YFP-PrP* NRK cells. Prior to starting the time-lapses, nuclei were stained with Hoechst. Image-collection was started immediately after the addition of thapsigargin (TG). Scale bar represents 20 µm. (D) Percentage of cells as represented in (C) with a perinuclear Golgi-localized pattern of YFP-PrP* after 30 min of TG treatment. The bar graph represents 3 biological replicates. Symbols were coded for each independently executed experiment. The number of cells analyzed for each biological replicate are as follows (CTL: triangle 29, diamond 56, circle 51; siTMED9: triangle 69, diamond 58, circle 61). Error bars represent standard deviation of the mean. Statistics were calculated from unpaired t-test with ** indicated p<0.01. (E) Immunofluorescence images of endogenous TMED9 in control siRNA or siTMED9-treated YFP-PrP* NRK cells that were depleted for TMED9. For each condition, cells were treated with thapsigargin (TG) for 0 min or 90 min before fixation and staining for TMED9 and ER-resident chaperone, calnexin (CNX). Nuclei were stained with Hoechst. Larger fields of views for these panels and additional data for these experiments are presented in . Scale bar represents 10 µm. (F) Plot of the average Pearson’s r values between YFP-PrP* and endogenous calnexin (CNX) for control (CTL) siRNA or siTMED-treated cells at 0 min or 90 min after thapsigargin addition, as shown in (E). The number of cells analyzed for each condition are listed as follows: CTL siRNA 0 min post-TG-treatment (n=14), CTL siRNA 90 min post-TG-treatment (n=12), siTMED9 0 min post-TG-treatment (n=15), siTMED9 90 min post-TG-treatment (n=14). Pearson’s colocalization coefficients, r, were measured between YFP-PrP* and CNX, within the boundaries of the cell as defined by the outline of the CNX staining. (G) Immunofluorescence images of endogenous TMED9 in control (CTL) or TMED9 siRNA (siTMED9)-treated YFP-PrP WT NRK cells that were depleted for TMED9. Cells were fixed and stained for TMED9 and ER-resident chaperone, calnexin (CNX) at steady-state conditions. Nuclei were stained with Hoechst. Scale bar represents 10 µm. (H) Plot of the average Pearson’s r values between YFP-PrP WT and endogenous calnexin (CNX) for control (CTL) siRNA or siTMED-treated cells at steady-state, as shown in (G). The number of cells analyzed for each condition are listed as follows: CTL siRNA (n=22) and siTMED9 (n=24). Pearson’s colocalization coefficients, r, were measured between YFP-PrP* and CNX, within the boundaries of the cell as defined by the outline of the CNX staining.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Western Blot, Control, Standard Deviation, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Representative western blots of YFP-PrP* NRK cells that were treated as listed below (n=3 biological replicates). Untreated (Control), treated with BRD4780 for 180 min (BRD), pretreated with bafilomycin A1 (BAF) for 180 min and co-treated with BRD for 180 min (BAF+BRD), pretreated with MG132 for 180 min and co-treated with BRD for 180 min (MG132+BRD), pretreated with 3-methyladenine (3MA) for 180 min and co-treated with BRD for 180 min (3MA+BRD) or pretreated with BFA for 180 min and co-treated with BRD for 180 min (BFA+BRD). Blots are probed for endogenous TMED9 and GAPDH. (B) Bar graph representing the mean band intensity for TMED9 from 3 biological replicates as shown in (A). For each condition, TMED9 band intensities were double normalized first by the band intensity of GAPDH and second within individual experiments using control band intensity. Orange bars represent the measurement for the low molecular weight (LMW) band of TMED9 and gray bars represent the measurement of the high molecular weight (HMW) band of TMED9. Error bars represent standard deviations of the mean. Symbols were coded for individual experiments. Statistics were calculated from unpaired t-test with Welch’s correction with * indicated p<0.05 and ** p<0.01. (C) Western blots of digested protein lysates of stably transfected YFP-PrP* NRK cells either in control condition (Control) or after a pretreatment of 2 h of bafilomycin followed by co-incubation with 2 h of BRD4780 (BAF+BRD). Protein lysates undigested (water) or digested with Endoglycosidase H (Endo H), Neuraminidase+O-Glycosidase (NA+O-Glyc.), Peptide-N-glycosidase F (PNG) or NA+O-Glyc+PNGase (NA+O-G+PNG) for 3 h at 37°C (n=1 experiment). Blots were probed for TMED9 and calnexin (CNX). (D) Western blot band graphics obtained by first analyzing the plot lanes and second generating the line graphs of pre-selected regions of interest (ROI) from the western blot for control samples (left) or BAF+BRD samples (right). Black arrow represents the HMW form, orange arrow represents the LMW and purple, the unglycosylated form of TMED9. (E) (left) Fluorescence image of a typical untreated YFP-PrP* NRK cell that was co-transfected with CER-TMED9 and ER-marker, mCherry-Sec61β. Scale bar represents 10 µm. (right) Magnified panel of the stroked region on the right. Scale bar represents 1 µm. (F) (left) Fluorescence image of a typical untreated YFP-PrP* NRK cell that was co-transfected with CER-TMED9 and Golgi marker, FusionRed-SiT. Scale bar represents 10 µm. (right) Magnified panel of the stroked region on the right. Scale bar represents 1 µm. (G) Time-lapse imaging sequence of a NRK cell that was co-transfected with CER-TMED9 and ER marker, mCherry-Sec61β. Image-collection was started immediately after the addition of 100 µM BRD4780 treatment. Scale bar is 10 µm. (H) Example of an ER mask created from the ER-marker, mCherry-Sec61β. The ER mask was used to measure the CER-TMED9 fluorescence intensity within the ER at 0 or 30 min time points after the addition of BRD4780 from 9 individual time-lapses as shown in (G) and quantified in (I). (I) Bar graph representing the mean of ER-localized CER-TMED9 fluorescence-intensity measurements of 9 individual time-lapses of CER-TMED9 and mCherry-Sec61β-expressing cells, as shown in (G). The ER mask was generated based on and as analyzed in in (H). Statistics were calculated from unpaired t-test with Welch’s correction with ** indicated p<0.01 (J) Representative immunofluorescence images of YFP-PrP* NRK cells that were transfected with CER-TMED9 under control untreated (CTL) or 100 µM BRD4780 (30min)-treated (BRD) conditions and stained for GM130. Scale bar is 20 µm. (K) Bar graph representing the mean of Pearson’s correlation coefficient r to measure colocalization between CER-TMED9 and GM130 under untreated (CTL) and BRD4780 (30 min)-treated (BRD) conditions, as exemplified in (J). Colocalization for 18 cells were measured for control conditions and 11 cells were measured for BRD4780 (30min)-treated cells. Statistics were calculated from unpaired t-test Welch’s correction with *** indicating p<0.001.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Western Blot, Control, Molecular Weight, High Molecular Weight, Stable Transfection, Transfection, Incubation, Fluorescence, Marker, Imaging, Sequencing, Expressing, Generated, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Western blots of digested lysates of YFP-PrP* NRK cells in control and BRD4780-treated conditions. Protein lysates were not digested (water) or digested with Endoglycosidase H (Endo H), Neuraminidase only (NA), Neuraminidase+O-Glycosidase (NA+O-G.), Peptide-N-glycosidase F (PNG) or Neuraminidase+O-Glycosidase + Peptide-N-glycosidase F (NA+O-G+PNG) for 3 h at 37°C (n=1 experiment). Blots were probed for TMED9 and calnexin (CNX). (B) Western blot band graphics were obtained by first analyzing the plot lanes and second by generating line graphs of pre-selected regions of interest (ROI) from the western blot for control samples (left) or BRD4780 (30 min)-treated samples (right). Black arrow represents the HMW form, orange arrow represents the LMW and purple, the unglycosylated form of TMED9.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Still frames of a typical YFP-PrP* NRK cell that was co-transfected with CER-TMED9 and mCherry-Sec61β and treated as follows. The first image is of the cell before treatment began (Untreated). The second image is of the cell after 30 min with 50 µg/ml of CHX + BRD4780 (CHX + BRD4780 30 min). The third image is of the cell 30 min after washing with CHX-only medium (CHX wash 30 min). The fourth image is of the cell 30 min after treatment with 50 µg/ml of CHX + 0.1 µM thapsigargin (CHX + TG 30 min). Juxtaposed with each image is an area zoom of the region within the stroked frame. Scale bar represents 10 µm in the larger field of view and in the magnified panel of the stroked region. These select images are also provided in Video 2. (B) Representative western blots of GFP-tag co-immunoprecipitations (co-IPs) from the parental untransfected NRK cells (P) or YFP-PrP* NRK cells (n=3 biological replicates). Quantified in (C). Cells were harvested at the indicated time points after BRD4780-treatment. Blots were probed for PrP to detect YFP-PrP*, or for endogenous calnexin (CNX) and TMED9. (C) Bar graph representing the mean band intensity for CNX and TMED9 in the eluates from 3 independently performed experiments as shown in (B). For each time point, CNX and TMED9 eluate band intensities were double normalized. First, they were normalized by the band intensity of the eluted YFP-PrP* band. Second, they were normalized by the time 0 eluate band intensity for each protein probed. Error bars represent standard deviations of the mean. Symbols were coded for individual experiments. Statistics were calculated from unpaired t-test with Welch’s correction with and ** indicated p<0.01. (D) Western blots of GFP-tag co-immunoprecipitations (co-IPs) from the parental untransfected NRK cells (P) or YFP-PrP* NRK cells (n=1 experiment). Cells were harvested after the indicated treatments. Blots were probed for PrP to detect YFP-PrP*, or for endogenous calnexin (CNX) and TMED9.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Transfection, Western Blot

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: Western blots of GFP-tag co-immunoprecipitations (co-IPs) from the parental untransfected NRK cells (P) or stably transfected YFP-PrP* NRK cells (n=1 experiment). YFP-PrP* NRK cells were treated with 100 µM BRD4780 (BRD) and collected for co-IP at the indicated time points. Cells were harvested at the indicated time points for co-immunoprecipitation of YFP-PrP* with anti-GFP antibody conjugated beads in addition to GFP to detect co-immunoprecipitation of YFP-PrP* constructs, blots were probed for endogenous calnexin (CNX), TMED2, TMP21, and TMED9.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Western Blot, Stable Transfection, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Construct

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) At steady-state, TMED9 (cyan), in conjunction with TMP21 (green), TMED2 (gold) and likely other export machinery (gray), such as other p24-family members, cargo receptors, COPII and COPI coat proteins (gray), traffic between the ER and Golgi, creating a pre-existing pathway for the ER-to-Golgi transport of select substrates. (B) Upon release from calnexin (red) during steady-state or ER-stress conditions, misfolded GPI-anchored proteins (GPI-APs) (depicted as purple circles) piggyback with p24-family members to access these p24-family populated ER-exit sites. (C) When TMED9 is depleted from the ER either by siRNA knockdown or by BRD4780-treatment, the TMED9 and p24-family populated ER-exit pathway collapses and misfolded GPI-APs are unable to exit the ER to the Golgi. Instead, they remain in association with calnexin. (B-C) Properly folded GPI-APs (depicted as purple triangles with yellow stripe) are able to exit the ER to the Golgi through alternate ER-exit sites for subsequent secretion regardless of the presence or absence of TMED9.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Knockdown