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Proteintech tmed10
Tmed10, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/tmed10/pm41932203-57-61-78?v=Proteintech
Average 93 stars, based on 22 article reviews

tmed10 - by Bioz Stars, 2026-08

93/100 stars

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(A and B) Expression level of endogenous TMEDs (TMED1-5 & 7, 9, 10) in wild-type (WT) or TMED10 knockout (10KO) HEK293T (A) and WT or 10KO HeLa cell lines (B) generated by CRISPR-Cas9. The endogenous TMED6 level was low and undetectable in these cell lines. The data are representative of three independent experiments. (C-L) Secretion of mIL-1α (C), mIL-33 (D), mIL-36α (E), mIL-36Ra (F), mIL-37 (G), Galectin-1 (H), Galectin-3 (I), Annexin A1 (J), HSPB5 (K) and Tau (L) in TMED10-KO HEK293T cells with control or expression of TMEDs (TMED1-7 & 9, 10). The data are representative of three independent experiments. (M) Level of endogenous and exogenous TMEDs in WT or TMED10-KO HEK293T cells with control or TMEDs (TMED1-7 & 9, 10) expression for 24h. (N) Secretion of mIL-1β in WT HEK293T cells with control or TMED10 (T10), LMAN2 (L2), ERGIC53 (E53) expression. The data are representative of three independent experiments. (O) Level of endogenous TMEDs (TMED1-5 & 7, 9, 10) in WT or TMED10-KO HEK293T cells with control or TMED10-V5 expression for 96h. (P) Surface delivery of CD59 analyzed by hook and release assay. WT and TMED10-KO HeLa cells with control or TMED10-V5 expression for 72 h were co-transfected with EGFP-SBP-CD59 and streptavidin-KDEL. 23 hours later, the cells were treated without or with 40 μM biotin for 1 h, and then immunofluorescence was conducted with anti-V5 and anti-GM130 antibody. Scale bar, 10 μm. (Q) Secretion of mIL-1β in WT HEK293T cells with control or TMEDs (TMED1-7 & 9, 10) expression. The data are representative of three independent experiments. (R) HEK-Blue IL-1β cells (InvivoGen) were treated with culture medium derived from indicated HEK293T cells or 0.1 μg/ml recombinant mIL-1β (rmIL-1β) as a positive control. Levels of SEAP indicating IL-1β activity in the medium were monitored using QUANTI-Blue (n=3).

Journal: bioRxiv

Article Title: TMEDs Mediate Versatile Cargo Transport in Vesicle-dependent Unconventional Secretion

doi: 10.1101/2025.05.04.652080

Figure Lengend Snippet: (A and B) Expression level of endogenous TMEDs (TMED1-5 & 7, 9, 10) in wild-type (WT) or TMED10 knockout (10KO) HEK293T (A) and WT or 10KO HeLa cell lines (B) generated by CRISPR-Cas9. The endogenous TMED6 level was low and undetectable in these cell lines. The data are representative of three independent experiments. (C-L) Secretion of mIL-1α (C), mIL-33 (D), mIL-36α (E), mIL-36Ra (F), mIL-37 (G), Galectin-1 (H), Galectin-3 (I), Annexin A1 (J), HSPB5 (K) and Tau (L) in TMED10-KO HEK293T cells with control or expression of TMEDs (TMED1-7 & 9, 10). The data are representative of three independent experiments. (M) Level of endogenous and exogenous TMEDs in WT or TMED10-KO HEK293T cells with control or TMEDs (TMED1-7 & 9, 10) expression for 24h. (N) Secretion of mIL-1β in WT HEK293T cells with control or TMED10 (T10), LMAN2 (L2), ERGIC53 (E53) expression. The data are representative of three independent experiments. (O) Level of endogenous TMEDs (TMED1-5 & 7, 9, 10) in WT or TMED10-KO HEK293T cells with control or TMED10-V5 expression for 96h. (P) Surface delivery of CD59 analyzed by hook and release assay. WT and TMED10-KO HeLa cells with control or TMED10-V5 expression for 72 h were co-transfected with EGFP-SBP-CD59 and streptavidin-KDEL. 23 hours later, the cells were treated without or with 40 μM biotin for 1 h, and then immunofluorescence was conducted with anti-V5 and anti-GM130 antibody. Scale bar, 10 μm. (Q) Secretion of mIL-1β in WT HEK293T cells with control or TMEDs (TMED1-7 & 9, 10) expression. The data are representative of three independent experiments. (R) HEK-Blue IL-1β cells (InvivoGen) were treated with culture medium derived from indicated HEK293T cells or 0.1 μg/ml recombinant mIL-1β (rmIL-1β) as a positive control. Levels of SEAP indicating IL-1β activity in the medium were monitored using QUANTI-Blue (n=3).

Article Snippet: We purchased mouse anti-HSP90AB1 antibody from Santa Cruz, rabbit anti-GRP94, anti-TMED2, anti-TMED3, anti-TMED4, anti-TMED9 and anti-TMED10 antibody from Proteintech, rabbit anti-TMED1, anti-TMED5, anti-TMED6, anti-TMED7 antibody from Novus Biologicals, cycloheximide (CHX), rabbit anti-V5, anti-HA and anti-GST antibody from CST, mouse anti-GM130 antibody from BD Biosciences, mouse anti-tubulin and anti-actin antibody from Abcam.

Techniques: Expressing, Knock-Out, Generated, CRISPR, Control, Release Assay, Transfection, Immunofluorescence, Derivative Assay, Recombinant, Positive Control, Activity Assay

(A) Secretion of mIL-1β in TMED10-KO HEK293T cells with control or TMEDs (TMED1-7 & 9, 10) expression. The data are representative of three independent experiments. (B) Heatmap showing the secretion of indicated cargoes in TMED10-KO HEK293T cells with control or TMEDs (TMED1-7 & 9, 10) expression as shown in (A) and . Relative level of cargo secretion was normalized to cargo level in cell lysates, and the TMED10 group was set as 1. (C) HEK-Blue IL-1β cells (InvivoGen) were treated with culture medium derived from TMED10-KO HEK293T cells with indicated protein expression, or with 0.1 μg/ml recombinant mIL-1β (rmIL-1β) as a positive control. Levels of SEAP indicating IL-1β activity in the medium were monitored using QUANTI-Blue (n = 3). (D) The quantitative secretomics workflow of U2OS cells with LMAN2-V5 or TMEDs-V5 (TMED1-5 & 7, 9, 10) stable expression. (E) Venn diagram depicting the overlap of upregulated cargoes identified in the secretome of TMEDs (TMED1-5 & 7, 9, 10) with proteins detected in human plasma. (F) Heatmap showing the secretome in U2OS cells with stable expression of LMAN2 or TMEDs (TMED1-5 & 7, 9, 10). Cargo secretion level in the LMAN2 group (control) was set as 1, and other sample groups were normalized relative to this control. The data are average of two independent experiments. (G) Relative level of indicated exosome markers and LDH release in the secretome of U2OS cells with LMAN2 or TMEDs (TMED1-5 & 7, 9, 10) stable expression. (H) Network diagram illustrating the relationships between different sets of upregulated UcPS cargoes of the TMEDs (TMED1-5 & 7, 9, 10). High-confidence upregulated UcPS cargoes unique to each TMED are arranged around the corresponding TMED, and shared cargoes are labeled with different colors denoting multiple combinations. (I) Heatmap showing the secretion of specific cargoes in U2OS cells with stable expression of LMAN2 or TMEDs (TMED1-7 & 9, 10), as measured by secretion assay (upper panel) shown in or mass spectrometry (lower panel). Relative level of cargo secretion was normalized to cargo expression in cell lysate and the group exhibiting the highest cargo secretion was set as 1. Blue boxes highlight the peak of specific TMED-enhanced secretion in both cellular secretion experiments and mass spectrometry.

Journal: bioRxiv

Article Title: TMEDs Mediate Versatile Cargo Transport in Vesicle-dependent Unconventional Secretion

doi: 10.1101/2025.05.04.652080

Figure Lengend Snippet: (A) Secretion of mIL-1β in TMED10-KO HEK293T cells with control or TMEDs (TMED1-7 & 9, 10) expression. The data are representative of three independent experiments. (B) Heatmap showing the secretion of indicated cargoes in TMED10-KO HEK293T cells with control or TMEDs (TMED1-7 & 9, 10) expression as shown in (A) and . Relative level of cargo secretion was normalized to cargo level in cell lysates, and the TMED10 group was set as 1. (C) HEK-Blue IL-1β cells (InvivoGen) were treated with culture medium derived from TMED10-KO HEK293T cells with indicated protein expression, or with 0.1 μg/ml recombinant mIL-1β (rmIL-1β) as a positive control. Levels of SEAP indicating IL-1β activity in the medium were monitored using QUANTI-Blue (n = 3). (D) The quantitative secretomics workflow of U2OS cells with LMAN2-V5 or TMEDs-V5 (TMED1-5 & 7, 9, 10) stable expression. (E) Venn diagram depicting the overlap of upregulated cargoes identified in the secretome of TMEDs (TMED1-5 & 7, 9, 10) with proteins detected in human plasma. (F) Heatmap showing the secretome in U2OS cells with stable expression of LMAN2 or TMEDs (TMED1-5 & 7, 9, 10). Cargo secretion level in the LMAN2 group (control) was set as 1, and other sample groups were normalized relative to this control. The data are average of two independent experiments. (G) Relative level of indicated exosome markers and LDH release in the secretome of U2OS cells with LMAN2 or TMEDs (TMED1-5 & 7, 9, 10) stable expression. (H) Network diagram illustrating the relationships between different sets of upregulated UcPS cargoes of the TMEDs (TMED1-5 & 7, 9, 10). High-confidence upregulated UcPS cargoes unique to each TMED are arranged around the corresponding TMED, and shared cargoes are labeled with different colors denoting multiple combinations. (I) Heatmap showing the secretion of specific cargoes in U2OS cells with stable expression of LMAN2 or TMEDs (TMED1-7 & 9, 10), as measured by secretion assay (upper panel) shown in or mass spectrometry (lower panel). Relative level of cargo secretion was normalized to cargo expression in cell lysate and the group exhibiting the highest cargo secretion was set as 1. Blue boxes highlight the peak of specific TMED-enhanced secretion in both cellular secretion experiments and mass spectrometry.

Techniques: Control, Expressing, Derivative Assay, Recombinant, Positive Control, Activity Assay, Clinical Proteomics, Labeling, Mass Spectrometry

(A) Immunofluorescence of U2OS cells co-expressing EGFP-ERGIC-53 and TMEDs-V5 with anti-V5 antibodies. Scale bar, 10 μm. (B) Schematic diagram of the RUSH system to retain ERGIC-localized TMEDs (TMED1-4 & 7, 9, 10) to the ER. TMEDs fused with a streptavidin binding peptide (SBP) are retained in the ER via binding to streptavidin (Str) connected to a KDEL sequence. Upon biotin addition, TMEDs were released and trafficked to ERGIC. (C) Immunofluorescence of TMED10-KO HeLa cells co-expressing EGFP-ERGIC-53 and SBP-TMED10-V5, without or with Str-KDEL expression, and treated without or with 40 μM biotin for 8 h. Scale bar, 10 μm. (D) Secretion assay of mIL-1β in TMED10-KO HEK293T cells combined with RUSH system. Cells were co-transfected without or with mIL-1β-FLAG, Str-KDEL, and SBP-TMED10-V5 and treated without or with biotin at the indicated concentration and time points. The data are representative of three independent experiments. (E) Schematic diagram of the RUSH system to retain ER-localized TMED5 to the ERGIC. TMED5 fused with SBP was retained via binding to streptavidin connected to TMED10ΔGOLD or LMAN2, two ERGIC-localized membrane protein deficient in UcPS. Upon biotin addition, TMED5 was released and trafficked back to the ER. (F) Immunofluorescence of TMED10-KO HeLa cells co-expressing EGFP-ERGIC-53 and SBP-TMED5-V5, without or with Str-TMED10ΔGOLD-HA expression, and treated without or with 40 μM biotin for 16 h. Scale bar, 10 μm. (G) Secretion assay of mIL-1β in TMED10-KO HEK293T cells combined with RUSH system. Cells were co-transfected without or with mIL-1β-FLAG, Str-TMED10ΔGOLD-HA, SBP-TMED5-V5 and treated without or with biotin at the indicated concentration and time points. The data are representative of three independent experiments. (H and I) Cell-free translocation assay of mIL-1β in TMED10-KO HEK293T cells combined with RUSH system. Cells were transfected without or with Str-KDEL and SBP-TMED10-V5 (H) or SBP-TMED2-V5 (I) and treated without or with 40 μM biotin for 16 h. The data are representative of three independent experiments. (J) Cell-free translocation assay of mIL-1β in TMED10-KO HEK293T cells combined with RUSH system. Cells were transfected without or with Str-TMED10ΔGOLD-HA and SBP-TMED5-V5 and treated without or with 40 μM biotin for 16 h. The data are representative of three independent experiments. (K) A model showing diversified UcPS cargo translocation mediated by TMEDs.

Journal: bioRxiv

Article Title: TMEDs Mediate Versatile Cargo Transport in Vesicle-dependent Unconventional Secretion

doi: 10.1101/2025.05.04.652080

Figure Lengend Snippet: (A) Immunofluorescence of U2OS cells co-expressing EGFP-ERGIC-53 and TMEDs-V5 with anti-V5 antibodies. Scale bar, 10 μm. (B) Schematic diagram of the RUSH system to retain ERGIC-localized TMEDs (TMED1-4 & 7, 9, 10) to the ER. TMEDs fused with a streptavidin binding peptide (SBP) are retained in the ER via binding to streptavidin (Str) connected to a KDEL sequence. Upon biotin addition, TMEDs were released and trafficked to ERGIC. (C) Immunofluorescence of TMED10-KO HeLa cells co-expressing EGFP-ERGIC-53 and SBP-TMED10-V5, without or with Str-KDEL expression, and treated without or with 40 μM biotin for 8 h. Scale bar, 10 μm. (D) Secretion assay of mIL-1β in TMED10-KO HEK293T cells combined with RUSH system. Cells were co-transfected without or with mIL-1β-FLAG, Str-KDEL, and SBP-TMED10-V5 and treated without or with biotin at the indicated concentration and time points. The data are representative of three independent experiments. (E) Schematic diagram of the RUSH system to retain ER-localized TMED5 to the ERGIC. TMED5 fused with SBP was retained via binding to streptavidin connected to TMED10ΔGOLD or LMAN2, two ERGIC-localized membrane protein deficient in UcPS. Upon biotin addition, TMED5 was released and trafficked back to the ER. (F) Immunofluorescence of TMED10-KO HeLa cells co-expressing EGFP-ERGIC-53 and SBP-TMED5-V5, without or with Str-TMED10ΔGOLD-HA expression, and treated without or with 40 μM biotin for 16 h. Scale bar, 10 μm. (G) Secretion assay of mIL-1β in TMED10-KO HEK293T cells combined with RUSH system. Cells were co-transfected without or with mIL-1β-FLAG, Str-TMED10ΔGOLD-HA, SBP-TMED5-V5 and treated without or with biotin at the indicated concentration and time points. The data are representative of three independent experiments. (H and I) Cell-free translocation assay of mIL-1β in TMED10-KO HEK293T cells combined with RUSH system. Cells were transfected without or with Str-KDEL and SBP-TMED10-V5 (H) or SBP-TMED2-V5 (I) and treated without or with 40 μM biotin for 16 h. The data are representative of three independent experiments. (J) Cell-free translocation assay of mIL-1β in TMED10-KO HEK293T cells combined with RUSH system. Cells were transfected without or with Str-TMED10ΔGOLD-HA and SBP-TMED5-V5 and treated without or with 40 μM biotin for 16 h. The data are representative of three independent experiments. (K) A model showing diversified UcPS cargo translocation mediated by TMEDs.

Techniques: Immunofluorescence, Expressing, Binding Assay, Sequencing, Transfection, Concentration Assay, Membrane, Translocation Assay

$(A) Secretion of FGF2 in TMED10-KO HEK293T cells without or with 10 μM punicalagin treatment for 1.5 h. The data are representative of three independent experiments. (B) Secretion of mIL-1β in TMED10-KO HEK293T cells with control or TMEDs (TMED1-7 & 9, 10) expression, without or with 10 μM punicalagin treatment for 1.5 h. The data are representative of three independent experiments. (C and D) Membrane floatation assay for the amounts of mIL-1β (C) and galectin-3 (D) in the membrane fractions and total cell lysates in TMED10-KO HEK293T cells with control or TMEDs-V5 (TMED1-7 & 9, 10) expression. (E and F) Quantification of the ratio of mIL-1β (E) and galectin-3 (F) (mean ± SEM) in membrane fractions as shown in (C) and (D), the control group was set as 1. p values were calculated by two-tailed t test (n ≥ 3). ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (G) Glycosylation detection in TMED10-KO HEK293T cells expressing TMEDs (TMED1-7 & 9, 10) or TMEDs glycosylation site mutants (TMED1-N53Q, TMED4-N117Q, TMED7-N103Q, and TMED9-N125Q) without or with PNGaseF digestion. The data are representative of three independent experiments. (H) Secretion of mIL-1β in TMED10-KO HEK293T cells with control or expression of TMEDs-V5 (TMED1, 4, 7, 9) or its glycosylation site mutant (TMED1-N53Q, TMED4-N117Q, TMED7-N103Q, and TMED9-N125Q). The data are representative of three independent experiments. (I) Coomassie blue staining of GST-TMEDs (TMED1-7 & 9, 10), HSP90AB1, HSP90B1, mIL-1β-FLAG, PCBD1 and RANGRF expressed in E.coli expression system and GST-TMEDs (TMED2 & 6) after thrombin digestion to remove GST-tag.$

Journal: bioRxiv

Article Title: TMEDs Mediate Versatile Cargo Transport in Vesicle-dependent Unconventional Secretion

doi: 10.1101/2025.05.04.652080

Figure Lengend Snippet: (A) Secretion of FGF2 in TMED10-KO HEK293T cells without or with 10 μM punicalagin treatment for 1.5 h. The data are representative of three independent experiments. (B) Secretion of mIL-1β in TMED10-KO HEK293T cells with control or TMEDs (TMED1-7 & 9, 10) expression, without or with 10 μM punicalagin treatment for 1.5 h. The data are representative of three independent experiments. (C and D) Membrane floatation assay for the amounts of mIL-1β (C) and galectin-3 (D) in the membrane fractions and total cell lysates in TMED10-KO HEK293T cells with control or TMEDs-V5 (TMED1-7 & 9, 10) expression. (E and F) Quantification of the ratio of mIL-1β (E) and galectin-3 (F) (mean ± SEM) in membrane fractions as shown in (C) and (D), the control group was set as 1. p values were calculated by two-tailed t test (n ≥ 3). ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (G) Glycosylation detection in TMED10-KO HEK293T cells expressing TMEDs (TMED1-7 & 9, 10) or TMEDs glycosylation site mutants (TMED1-N53Q, TMED4-N117Q, TMED7-N103Q, and TMED9-N125Q) without or with PNGaseF digestion. The data are representative of three independent experiments. (H) Secretion of mIL-1β in TMED10-KO HEK293T cells with control or expression of TMEDs-V5 (TMED1, 4, 7, 9) or its glycosylation site mutant (TMED1-N53Q, TMED4-N117Q, TMED7-N103Q, and TMED9-N125Q). The data are representative of three independent experiments. (I) Coomassie blue staining of GST-TMEDs (TMED1-7 & 9, 10), HSP90AB1, HSP90B1, mIL-1β-FLAG, PCBD1 and RANGRF expressed in E.coli expression system and GST-TMEDs (TMED2 & 6) after thrombin digestion to remove GST-tag.

Techniques: Control, Expressing, Membrane, Two Tailed Test, Glycoproteomics, Mutagenesis, Staining

(A) Schematic diagram of the cell-free translocation assay. Briefly, membranes from TMED10-KO HEK293T cells with control or TMEDs-V5 (TMED1-7 & 9, 10) expression were incubated with cargo, cytosol, ATPR & GTP to generate the cell-free translocation reaction. Membrane flotation and proteinase K digestion were then performed to determine the amount of membrane-incorporated cargo. ATPR: ATP regeneration system. (B) Cell-free translocation of mIL-1β using membranes from TMED10-KO HEK293T cells without or with TMEDs (TMED4, 7, 10) expression. (C) Quantification of the translocation efficiency of mIL-1β (mean ± SEM) without or with TMEDs (TMED1-7 & 9, 10) expression as shown in (B) and , the relative level of mIL-1β translocation was normalized to the control group; p values were calculated by one-way analysis of variance (ANOVA) (n ≥ 2). (D) Cell-free translocation of RANGRF using membranes from TMED10-KO HEK293T cells without or with TMEDs (TMED6, 9, 10) expression. (E) Quantification of the translocation efficiency of RANGRF (mean ± SEM) without or with TMEDs (TMED1-7 & 9, 10) expression as shown in (D) and , the relative level of RANGRF translocation was normalized to the TMED9 group; p values were calculated by one-way ANOVA (n ≥ 3). (F) Cell-free translocation of PCBD1 using membranes from TMED10-KO HEK293T cells with TMEDs (TMED1-3 & 10) expression. (G) Quantification of the translocation efficiency of PCBD1 (mean ± SEM) without or with TMEDs (TMED1-7 & 9, 10) expression as shown in (F) and ( , F and G), the relative level of PCBD1 translocation was normalized to the TMED10 group; p values were calculated by one-way ANOVA (n ≥ 3). ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Journal: bioRxiv

Article Title: TMEDs Mediate Versatile Cargo Transport in Vesicle-dependent Unconventional Secretion

doi: 10.1101/2025.05.04.652080

Figure Lengend Snippet: (A) Schematic diagram of the cell-free translocation assay. Briefly, membranes from TMED10-KO HEK293T cells with control or TMEDs-V5 (TMED1-7 & 9, 10) expression were incubated with cargo, cytosol, ATPR & GTP to generate the cell-free translocation reaction. Membrane flotation and proteinase K digestion were then performed to determine the amount of membrane-incorporated cargo. ATPR: ATP regeneration system. (B) Cell-free translocation of mIL-1β using membranes from TMED10-KO HEK293T cells without or with TMEDs (TMED4, 7, 10) expression. (C) Quantification of the translocation efficiency of mIL-1β (mean ± SEM) without or with TMEDs (TMED1-7 & 9, 10) expression as shown in (B) and , the relative level of mIL-1β translocation was normalized to the control group; p values were calculated by one-way analysis of variance (ANOVA) (n ≥ 2). (D) Cell-free translocation of RANGRF using membranes from TMED10-KO HEK293T cells without or with TMEDs (TMED6, 9, 10) expression. (E) Quantification of the translocation efficiency of RANGRF (mean ± SEM) without or with TMEDs (TMED1-7 & 9, 10) expression as shown in (D) and , the relative level of RANGRF translocation was normalized to the TMED9 group; p values were calculated by one-way ANOVA (n ≥ 3). (F) Cell-free translocation of PCBD1 using membranes from TMED10-KO HEK293T cells with TMEDs (TMED1-3 & 10) expression. (G) Quantification of the translocation efficiency of PCBD1 (mean ± SEM) without or with TMEDs (TMED1-7 & 9, 10) expression as shown in (F) and ( , F and G), the relative level of PCBD1 translocation was normalized to the TMED10 group; p values were calculated by one-way ANOVA (n ≥ 3). ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Techniques: Translocation Assay, Control, Expressing, Incubation, Membrane

(A) Cell-free translocation of mIL-1β using membranes from TMED10-KO HEK293T cells without or with TMED10-V5 expression, without or with ATPR and GTP treatment. The data are representative of three independent experiments. (B and C) Cell-free translocation of mIL-1β using membranes from TMED10-KO HEK293T cells without or with TMEDs (TMED1, 3, 5 for (B), TMED2, 6, 9 for (C)) expression. (D and E) Cell-free translocation of RANGRF using membranes from TMED10-KO HEK293T cells with TMEDs (TMED1, 2, 3, 9 for (D), TMED4, 5, 7, 9 for (E)) expression. (F and G) Cell-free translocation of PCBD1 using membranes from TMED10-KO HEK293T cells without or with TMEDs (TMED6, 9, 10 for (F) TMED4, 5, 7, 10 for (G)) expression.

Journal: bioRxiv

Article Title: TMEDs Mediate Versatile Cargo Transport in Vesicle-dependent Unconventional Secretion

doi: 10.1101/2025.05.04.652080

Figure Lengend Snippet: (A) Cell-free translocation of mIL-1β using membranes from TMED10-KO HEK293T cells without or with TMED10-V5 expression, without or with ATPR and GTP treatment. The data are representative of three independent experiments. (B and C) Cell-free translocation of mIL-1β using membranes from TMED10-KO HEK293T cells without or with TMEDs (TMED1, 3, 5 for (B), TMED2, 6, 9 for (C)) expression. (D and E) Cell-free translocation of RANGRF using membranes from TMED10-KO HEK293T cells with TMEDs (TMED1, 2, 3, 9 for (D), TMED4, 5, 7, 9 for (E)) expression. (F and G) Cell-free translocation of PCBD1 using membranes from TMED10-KO HEK293T cells without or with TMEDs (TMED6, 9, 10 for (F) TMED4, 5, 7, 10 for (G)) expression.

Techniques: Translocation Assay, Expressing

(A and B) Duolink PLA assay performed with HeLa cells expressing empty vector (Ctr), TMED9-V5 (T9), TMED10-V5 (T10), TMED10-TMED9CT (cytoplasmic tail, T10-9CT), TMED9ΔCT (T9ΔCT), TMED9-TMED10CT (T9-10CT) respectively and RANGRF-FLAG to test their interactions (A). The puncta of duolink signal area per cell (mean ± SEM) was quantified in (B); p values were calculated by one-way ANOVA (> 50 cells from three independent experiments). Scale bar, 10 μm. (C and D) Duolink PLA assay performed with HeLa cells expressing empty vector (Ctr), TMED9-V5 (T9), TMED10-V5 (T10), TMED9-TMED10CT(T9-10CT), TMED10ΔCT (T10ΔCT), TMED10-TMED9CT (T10-9CT) respectively and PCBD1-FLAG to test their interactions (C). The puncta of duolink signal area per cell (mean ± SEM) was quantified in (D); p values were calculated by one-way ANOVA (> 50 cells from three independent experiments). Scale bar, 10 μm. (E and F) Secretion of RANGRF (E) or PCBD1 (F) in TMED10-KO HEK293T cells with control or indicated TMED variants expression. The data are representative of three independent experiments. (G and H) Cell-free translocation of RANGRF (G) or PCBD1 (H) using membranes from TMED10-KO HEK293T cells without or with indicated TMED variants expression. The data are representative of three independent experiments. (I) Duolink PLA assay performed with HeLa cells expressing empty vector (Ctr), PCBD1 wild type (WT), or disease-associated mutations R88Q and Q98* respectively and TMED10-V5 to test their interaction. Scale bar, 10 μm. (J) Quantification of the puncta of duolink signal area per cell (mean ± SEM) as shown in (I); p values were calculated by one-way ANOVA (> 50 cells from three independent experiments). (K) Secretion of PCBD1 WT, disease-associated mutations R88Q and Q98* in TMED10-KO HEK293T cells with control or TMED10-V5 expression. (L) Quantification of the PCBD1 WT, disease-associated mutations R88Q and Q98* secretion (mean ± SEM) as shown in (K); p values were calculated by one-way ANOVA (n = 5). (M) Surface overview showing multiple charge interactions and hydrogen bonds between PCBD1 and T10-CT (TMED10 cytoplasmic tail) which was predicted by AlphaFold 3. (N) Upper, Ribbon diagram of the interaction between PCBD1 and T10-CT. PCBD1 is colored in pink and T10-CT is colored in cyan. Lower, two close-up views of the ribbon diagram showing the charge interactions of PCBD1-R88 with TMED10-E219, and hydrogen bond between PCBD1-Q98 and TMED10-R209. ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Journal: bioRxiv

Article Title: TMEDs Mediate Versatile Cargo Transport in Vesicle-dependent Unconventional Secretion

doi: 10.1101/2025.05.04.652080

Figure Lengend Snippet: (A and B) Duolink PLA assay performed with HeLa cells expressing empty vector (Ctr), TMED9-V5 (T9), TMED10-V5 (T10), TMED10-TMED9CT (cytoplasmic tail, T10-9CT), TMED9ΔCT (T9ΔCT), TMED9-TMED10CT (T9-10CT) respectively and RANGRF-FLAG to test their interactions (A). The puncta of duolink signal area per cell (mean ± SEM) was quantified in (B); p values were calculated by one-way ANOVA (> 50 cells from three independent experiments). Scale bar, 10 μm. (C and D) Duolink PLA assay performed with HeLa cells expressing empty vector (Ctr), TMED9-V5 (T9), TMED10-V5 (T10), TMED9-TMED10CT(T9-10CT), TMED10ΔCT (T10ΔCT), TMED10-TMED9CT (T10-9CT) respectively and PCBD1-FLAG to test their interactions (C). The puncta of duolink signal area per cell (mean ± SEM) was quantified in (D); p values were calculated by one-way ANOVA (> 50 cells from three independent experiments). Scale bar, 10 μm. (E and F) Secretion of RANGRF (E) or PCBD1 (F) in TMED10-KO HEK293T cells with control or indicated TMED variants expression. The data are representative of three independent experiments. (G and H) Cell-free translocation of RANGRF (G) or PCBD1 (H) using membranes from TMED10-KO HEK293T cells without or with indicated TMED variants expression. The data are representative of three independent experiments. (I) Duolink PLA assay performed with HeLa cells expressing empty vector (Ctr), PCBD1 wild type (WT), or disease-associated mutations R88Q and Q98* respectively and TMED10-V5 to test their interaction. Scale bar, 10 μm. (J) Quantification of the puncta of duolink signal area per cell (mean ± SEM) as shown in (I); p values were calculated by one-way ANOVA (> 50 cells from three independent experiments). (K) Secretion of PCBD1 WT, disease-associated mutations R88Q and Q98* in TMED10-KO HEK293T cells with control or TMED10-V5 expression. (L) Quantification of the PCBD1 WT, disease-associated mutations R88Q and Q98* secretion (mean ± SEM) as shown in (K); p values were calculated by one-way ANOVA (n = 5). (M) Surface overview showing multiple charge interactions and hydrogen bonds between PCBD1 and T10-CT (TMED10 cytoplasmic tail) which was predicted by AlphaFold 3. (N) Upper, Ribbon diagram of the interaction between PCBD1 and T10-CT. PCBD1 is colored in pink and T10-CT is colored in cyan. Lower, two close-up views of the ribbon diagram showing the charge interactions of PCBD1-R88 with TMED10-E219, and hydrogen bond between PCBD1-Q98 and TMED10-R209. ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Techniques: Expressing, Plasmid Preparation, Control, Translocation Assay

(A, C, E, G, I and K) TMED10-KO HeLa cells were co-expressed EGFP-ERGIC-53 and SBP-TMEDs-V5 (SBP-TMED1-4 & 7, 9-V5), without or with Str-KDEL expression, and treated without or with 40 μM biotin for 4 h. Immunofluorescence was performed with anti-V5 antibody. Scale bar, 10 μm. (B, D, F, H, J and L) Secretion assay of mIL-1β in TMED10-KO HEK293T cells combined with RUSH system. The cells were co-transfected with mIL-1β-FLAG, Str-KDEL, and SBP-TMEDs-V5 (SBP-TMED1-4 & 7, 9-V5) as indicated and treated without or with biotin at the indicated concentration and time points. The data are representative of three independent experiments. (M) TMED10-KO HeLa cells were co-expressed EGFP-ERGIC-53 and SBP-TMED5-V5, without or with Str-LMAN2-HA expression, and treated without or with 40 μM biotin for 16 h. Immunofluorescence was performed with anti-V5 and anti-HA antibody. Scale bar, 10 μm. (N) Secretion assay of mIL-1β in TMED10-KO HEK293T cells combined with RUSH system. The cells were co-transfected with mIL-1β-FLAG, Str-LMAN2-HA, and SBP-TMED5-V5 as indicated and treated without or with 40 μM biotin for 16 h. The data are representative of three independent experiments. (O) Secretion assay in HEK293T cells with control or TMED10-V5, and with conventional secretion cargo ss-mIL-1β-O-motif or UcPS cargo mIL-1β-O-motif expression (ss for signal peptide, O-motif for a reported O-GalNAc glycosylation motif with amino acid sequence GATGAGAGAGTTPGPG). The data are representative of three independent experiments. (P) Secretion and O-glycoprotease digestion assay in HEK293T cells with control or TMED10-V5, and with ss-mIL-1β-O-motif or mIL-1β-O-motif expression. The medium was collected and treated without or with O-glycoprotease IMPa (NEB). The data are representative of three independent experiments.

Journal: bioRxiv

Article Title: TMEDs Mediate Versatile Cargo Transport in Vesicle-dependent Unconventional Secretion

doi: 10.1101/2025.05.04.652080

Figure Lengend Snippet: (A, C, E, G, I and K) TMED10-KO HeLa cells were co-expressed EGFP-ERGIC-53 and SBP-TMEDs-V5 (SBP-TMED1-4 & 7, 9-V5), without or with Str-KDEL expression, and treated without or with 40 μM biotin for 4 h. Immunofluorescence was performed with anti-V5 antibody. Scale bar, 10 μm. (B, D, F, H, J and L) Secretion assay of mIL-1β in TMED10-KO HEK293T cells combined with RUSH system. The cells were co-transfected with mIL-1β-FLAG, Str-KDEL, and SBP-TMEDs-V5 (SBP-TMED1-4 & 7, 9-V5) as indicated and treated without or with biotin at the indicated concentration and time points. The data are representative of three independent experiments. (M) TMED10-KO HeLa cells were co-expressed EGFP-ERGIC-53 and SBP-TMED5-V5, without or with Str-LMAN2-HA expression, and treated without or with 40 μM biotin for 16 h. Immunofluorescence was performed with anti-V5 and anti-HA antibody. Scale bar, 10 μm. (N) Secretion assay of mIL-1β in TMED10-KO HEK293T cells combined with RUSH system. The cells were co-transfected with mIL-1β-FLAG, Str-LMAN2-HA, and SBP-TMED5-V5 as indicated and treated without or with 40 μM biotin for 16 h. The data are representative of three independent experiments. (O) Secretion assay in HEK293T cells with control or TMED10-V5, and with conventional secretion cargo ss-mIL-1β-O-motif or UcPS cargo mIL-1β-O-motif expression (ss for signal peptide, O-motif for a reported O-GalNAc glycosylation motif with amino acid sequence GATGAGAGAGTTPGPG). The data are representative of three independent experiments. (P) Secretion and O-glycoprotease digestion assay in HEK293T cells with control or TMED10-V5, and with ss-mIL-1β-O-motif or mIL-1β-O-motif expression. The medium was collected and treated without or with O-glycoprotease IMPa (NEB). The data are representative of three independent experiments.

Techniques: Expressing, Immunofluorescence, Transfection, Concentration Assay, Control, Glycoproteomics, Sequencing

(A) Label-free mass spectrometry quantification showing the relative amount of endogenous TMEDs (TMED1-5 & 7, 9, 10) in HeLa and U2OS cells with two replicates respectively. TMED6 was undetectable in this two cell lines. (B and C) Quantification of the relative abundance of four TMED subfamilies in HeLa (B) and U2OS (C) cells as shown in (A). (D) Heatmap showing the relative abundance of TMEDs (TMED1-5 & 7, 9, 10) and four subfamilies in human cell lines from multiple tissues. (E) Gel filtration assay analyzing the endogenous TMEDs (TMED1-5 & 7, 9, 10). HeLa cells were lysed and then applied to a Superdex 200 HR 10/30 column in 1% β-OG buffer. (F and H) Secretion of RANGRF (F) or PCBD1 (H) in TMED10-KO HEK293T cells with control or individual expression of four TMED subfamily members (TMED2, 5, 9, 10) or simultaneous expression to form hetero-tetramer. (G and I) Quantification of the RANGRF (G) or PCBD1 (I) secretion (mean ± SEM) as shown in (F) and (H); p values were calculated by one-way ANOVA (n = 3). (J) Cell-free translocation of RANGRF using membranes from TMED10-KO HEK293T cells expressing TMED9 alone or with three other subfamily members (TMED2, 5, 10) co-expression. (K) Quantification of the translocation efficiency of RANGRF (mean ± SEM) as shown in (J), the relative level of RANGRF translocation was normalized to the TMED9 group; p values were calculated by one-way ANOVA (n = 3). (L) Cell-free translocation of PCBD1 using membranes from TMED10-KO HEK293T cells expressing TMED10 alone or with three other subfamily members (TMED2, 5, 9) co-expression. (M) Quantification of the translocation efficiency of PCBD1 (mean ± SEM) as shown in (L), the relative level of PCBD1 translocation was normalized to the TMED10 group; p values were calculated by one-way ANOVA (n = 3). ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Journal: bioRxiv

Article Title: TMEDs Mediate Versatile Cargo Transport in Vesicle-dependent Unconventional Secretion

doi: 10.1101/2025.05.04.652080

Figure Lengend Snippet: (A) Label-free mass spectrometry quantification showing the relative amount of endogenous TMEDs (TMED1-5 & 7, 9, 10) in HeLa and U2OS cells with two replicates respectively. TMED6 was undetectable in this two cell lines. (B and C) Quantification of the relative abundance of four TMED subfamilies in HeLa (B) and U2OS (C) cells as shown in (A). (D) Heatmap showing the relative abundance of TMEDs (TMED1-5 & 7, 9, 10) and four subfamilies in human cell lines from multiple tissues. (E) Gel filtration assay analyzing the endogenous TMEDs (TMED1-5 & 7, 9, 10). HeLa cells were lysed and then applied to a Superdex 200 HR 10/30 column in 1% β-OG buffer. (F and H) Secretion of RANGRF (F) or PCBD1 (H) in TMED10-KO HEK293T cells with control or individual expression of four TMED subfamily members (TMED2, 5, 9, 10) or simultaneous expression to form hetero-tetramer. (G and I) Quantification of the RANGRF (G) or PCBD1 (I) secretion (mean ± SEM) as shown in (F) and (H); p values were calculated by one-way ANOVA (n = 3). (J) Cell-free translocation of RANGRF using membranes from TMED10-KO HEK293T cells expressing TMED9 alone or with three other subfamily members (TMED2, 5, 10) co-expression. (K) Quantification of the translocation efficiency of RANGRF (mean ± SEM) as shown in (J), the relative level of RANGRF translocation was normalized to the TMED9 group; p values were calculated by one-way ANOVA (n = 3). (L) Cell-free translocation of PCBD1 using membranes from TMED10-KO HEK293T cells expressing TMED10 alone or with three other subfamily members (TMED2, 5, 9) co-expression. (M) Quantification of the translocation efficiency of PCBD1 (mean ± SEM) as shown in (L), the relative level of PCBD1 translocation was normalized to the TMED10 group; p values were calculated by one-way ANOVA (n = 3). ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Techniques: Mass Spectrometry, Filtration, Control, Expressing, Translocation Assay

$(A) Schematic diagram of membrane fractionation using differential centrifugation and sucrose gradient centrifugation to separate ERGIC-enriched L fraction and ER-enriched P fraction followed by co-immunoprecipitation. (B) Co-IP (Co-immunoprecipitation) assay performed with the ERGIC-enriched fraction (L) and the ER-enriched fraction (P) extracted from HEK293T cells expressing HA-TMED10, without or with TMED10-V5 co-expression using anti-V5 agarose. (C) Quantification of the association efficiency of TMED10-V5 with other subfamily members (TMED2, 5, 9) or HA-TMED10 (mean ± SEM) as shown in (B); p values were calculated by two-way ANOVA (n = 3). (D) Co-IP assay was performed with HEK293T cells co-expressing TMED10-V5 and HA-TMED10 without or with GFP-Rab1A (R1A) or GFP-Rab1B (R1B) using anti-V5 agarose. (E) Quantification of the association efficiency of TMED10-V5 with other subfamily members (TMED2, 5, 9) or HA-TMED10 (mean ± SEM) as shown in (D); p values were calculated by two-way ANOVA (n = 3). (F) Turnover of TMED10-V5 in CHX chase assay without or with mIL-1β-FLAG or its UcPS-deficient mutant mIL-1β-WY-LL or three other subfamily members (HA-TMED2, 5, 9) expression in TMED10 KO HeLa cells. (G) Quantification of normalized TMED10-V5 (mean ± SEM) as shown in (F), the relative level of TMED10-V5 was normalized to Tubulin and the 0 h control group was set as 1; p values were calculated by two-way ANOVA (n ≥ 3). (H) Turnover of TMED10-V5 in CHX chase assay without or with mIL-36α, mIL-36α-F10A and HA-TMED2, 5, 9 expression in TMED10 KO HeLa cells. (I) Quantification of normalized TMED10-V5 (mean ± SEM) as shown in (H); p values were calculated by two-way ANOVA (n ≥ 2). (J) Turnover of TMED10-V5 in CHX chase assay without or with RAB1A, RAB1A UcPS-deficient mutant T75D and HA-TMED2, 5, 9 expression in TMED10 KO HEK293T cells. (K) Quantification of normalized TMED10-V5 (mean ± SEM) as shown in (J); p values were calculated by two-way ANOVA (n = 4). (L) Turnover of TMED10-V5 in CHX chase assay without or with RAB1B, RAB1B UcPS-deficient mutant T72D and HA-TMED2, 5, 9 expression in TMED10 KO HEK293T cells. (M) Quantification of normalized TMED10-V5 (mean ± SEM) as shown in (L); p values were calculated by two-way ANOVA (n = 4). (N) Model showing two distinct populations of TMED oligomers exist in cellular trafficking pathway. TMED hetero-oligomers play a role in conventional cargo (e.g. GPI-anchored proteins) transport between ER and ERGIC, while TMED homo-oligomers mediate unconventional secretion cargo translocation at the ERGIC. UcPS cargo stabilizes the homo-oligomeric structures, which subsequently facilitates translocation. ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.$

Journal: bioRxiv

Article Title: TMEDs Mediate Versatile Cargo Transport in Vesicle-dependent Unconventional Secretion

doi: 10.1101/2025.05.04.652080

Figure Lengend Snippet: (A) Schematic diagram of membrane fractionation using differential centrifugation and sucrose gradient centrifugation to separate ERGIC-enriched L fraction and ER-enriched P fraction followed by co-immunoprecipitation. (B) Co-IP (Co-immunoprecipitation) assay performed with the ERGIC-enriched fraction (L) and the ER-enriched fraction (P) extracted from HEK293T cells expressing HA-TMED10, without or with TMED10-V5 co-expression using anti-V5 agarose. (C) Quantification of the association efficiency of TMED10-V5 with other subfamily members (TMED2, 5, 9) or HA-TMED10 (mean ± SEM) as shown in (B); p values were calculated by two-way ANOVA (n = 3). (D) Co-IP assay was performed with HEK293T cells co-expressing TMED10-V5 and HA-TMED10 without or with GFP-Rab1A (R1A) or GFP-Rab1B (R1B) using anti-V5 agarose. (E) Quantification of the association efficiency of TMED10-V5 with other subfamily members (TMED2, 5, 9) or HA-TMED10 (mean ± SEM) as shown in (D); p values were calculated by two-way ANOVA (n = 3). (F) Turnover of TMED10-V5 in CHX chase assay without or with mIL-1β-FLAG or its UcPS-deficient mutant mIL-1β-WY-LL or three other subfamily members (HA-TMED2, 5, 9) expression in TMED10 KO HeLa cells. (G) Quantification of normalized TMED10-V5 (mean ± SEM) as shown in (F), the relative level of TMED10-V5 was normalized to Tubulin and the 0 h control group was set as 1; p values were calculated by two-way ANOVA (n ≥ 3). (H) Turnover of TMED10-V5 in CHX chase assay without or with mIL-36α, mIL-36α-F10A and HA-TMED2, 5, 9 expression in TMED10 KO HeLa cells. (I) Quantification of normalized TMED10-V5 (mean ± SEM) as shown in (H); p values were calculated by two-way ANOVA (n ≥ 2). (J) Turnover of TMED10-V5 in CHX chase assay without or with RAB1A, RAB1A UcPS-deficient mutant T75D and HA-TMED2, 5, 9 expression in TMED10 KO HEK293T cells. (K) Quantification of normalized TMED10-V5 (mean ± SEM) as shown in (J); p values were calculated by two-way ANOVA (n = 4). (L) Turnover of TMED10-V5 in CHX chase assay without or with RAB1B, RAB1B UcPS-deficient mutant T72D and HA-TMED2, 5, 9 expression in TMED10 KO HEK293T cells. (M) Quantification of normalized TMED10-V5 (mean ± SEM) as shown in (L); p values were calculated by two-way ANOVA (n = 4). (N) Model showing two distinct populations of TMED oligomers exist in cellular trafficking pathway. TMED hetero-oligomers play a role in conventional cargo (e.g. GPI-anchored proteins) transport between ER and ERGIC, while TMED homo-oligomers mediate unconventional secretion cargo translocation at the ERGIC. UcPS cargo stabilizes the homo-oligomeric structures, which subsequently facilitates translocation. ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Techniques: Membrane, Fractionation, Centrifugation, Gradient Centrifugation, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Mutagenesis, Control, Translocation Assay

(A and B) Secretion of wild-type mIL-1β (W) & mIL-1β-WY (9,10)-LL mutant (M), or wild-type mIL-36α (W) & mIL-36α-F10A (the site in the motif-1 identified previously) mutant (M) in TMED10-KO HEK293T cells with expression of TMEDs (TMED1, 2, 4, 7, 9, 10). The data are representative of three independent experiments. (C) Heatmap showing the relative secretion of mIL-1β & mIL-1β WY (9,10)-LL, and mIL36α & mIL36α-F10A in (A and B). (D) Turnover of TMED10-V5 in CHX chase assay without or with mIL-1β-FLAG, or its UcPS-deficient mutant (WY-LL) or three other subfamily members (HA-TMED2, 5, 9) co-expression in TMED10 KO HEK293T cells. (E) Quantification of normalized TMED10-V5 (mean ± SEM) as shown in (D), the relative level of TMED10-V5 was normalized to Tubulin and the 0 h control group was set as 1; p values were calculated by two-way ANOVA (n ≥ 3). (F) Turnover of TMED10-V5 in CHX chase assay without or with mIL-36α, mIL-36α-F10A and HA-TMED2, 5, 9 co-expression in TMED10 KO HEK293T cells. (G) Quantification of normalized TMED10-V5 (mean ± SEM) as shown in (F); p values were calculated by two-way ANOVA (n ≥ 2). ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Journal: bioRxiv

Article Title: TMEDs Mediate Versatile Cargo Transport in Vesicle-dependent Unconventional Secretion

doi: 10.1101/2025.05.04.652080

Figure Lengend Snippet: (A and B) Secretion of wild-type mIL-1β (W) & mIL-1β-WY (9,10)-LL mutant (M), or wild-type mIL-36α (W) & mIL-36α-F10A (the site in the motif-1 identified previously) mutant (M) in TMED10-KO HEK293T cells with expression of TMEDs (TMED1, 2, 4, 7, 9, 10). The data are representative of three independent experiments. (C) Heatmap showing the relative secretion of mIL-1β & mIL-1β WY (9,10)-LL, and mIL36α & mIL36α-F10A in (A and B). (D) Turnover of TMED10-V5 in CHX chase assay without or with mIL-1β-FLAG, or its UcPS-deficient mutant (WY-LL) or three other subfamily members (HA-TMED2, 5, 9) co-expression in TMED10 KO HEK293T cells. (E) Quantification of normalized TMED10-V5 (mean ± SEM) as shown in (D), the relative level of TMED10-V5 was normalized to Tubulin and the 0 h control group was set as 1; p values were calculated by two-way ANOVA (n ≥ 3). (F) Turnover of TMED10-V5 in CHX chase assay without or with mIL-36α, mIL-36α-F10A and HA-TMED2, 5, 9 co-expression in TMED10 KO HEK293T cells. (G) Quantification of normalized TMED10-V5 (mean ± SEM) as shown in (F); p values were calculated by two-way ANOVA (n ≥ 2). ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Techniques: Mutagenesis, Expressing, Control

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