Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: The CD30 ligand isoforms have distinct C-terminal extracellular regions. ( A ) Protein alignment of the two isoforms using Clustal Omega. Red—shared intracellular domain; cyan—shared transmembrane domain; purple—shared extracellular domain; black—predicted alpha helix within shared extracellular domain; yellow—extracellular sequences unique to each isoform. Asterisks indicate matching amino acid identity, dots and double dots indicate similarity, and hyphen indicates absence of residues in Iso2. The N-terminus for both proteins is intracellular and identical through residue 136, which includes the transmembrane domain at residues 38–62. The residues following 136 are distinct extracellular C-termini. ( B ) The predicted structural homology of CD30L isoforms to known protein structures generated by the Phyre2 web portal. Iso1 ( left ) showed strong homology to human TNF-alpha with several antiparallel beta-sheets. However, the software did not predict the presence of strong secondary structures for Iso2 ( right ), instead matching homology to small portions of proteins with unstructured domains. The two isoforms did share a predicted alpha helix that was membrane proximal on the extracellular side, present on the left side of each image.

Article Snippet: Clones were tested and chosen for response to CD30L-Fc recombinant protein (Sino Biological 10040-H01H, Wayne, PA, USA) via luciferase assay (described below).

Techniques: Residue, Generated, Software, Membrane

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Both CD30L isoforms were expressed at the RNA level in primary human cells. ( A ) PBMCs were isolated from healthy donors ( n = 6). Cells were either untreated (nt) or stimulated with DMSO, PMA/ionomycin (P/I), lipopolysaccharide (LPS) isolated from E . coli 0111:B4 (LPS), anti-CD3/CD28 beads (CD3/28), or immune complex (IC) for 6 or 24 h. The graphs show actin-normalized expression of both isoforms at both timepoints for all stimulation conditions. Single asterisks indicate p ≤ 0.05 and double asterisks indicate p ≤ 0.01 between the conditions indicated. Error bars represent standard deviation between donors. ( B ) PBMCs were isolated from healthy donors ( n = 3, half of the 6 total donors). Cells were either untreated (nt) or stimulated with PMA/ionomycin (P/I) or immune complex (IC) for 24 h. After incubation, cells were subject to flow cytometry for B cell marker CD19, monocyte marker CD14, and T cell marker CD3, as well as CD30 ligand Iso1. PBMC cell types were present at expected ratios. Error bars are standard deviation between donors.

Article Snippet: Clones were tested and chosen for response to CD30L-Fc recombinant protein (Sino Biological 10040-H01H, Wayne, PA, USA) via luciferase assay (described below).

Techniques: Isolation, Expressing, Standard Deviation, Incubation, Flow Cytometry, Marker

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 has a significantly impaired ability to bind CD30 compared to Iso1, but both are present in an exogenous CD30 protein complex. ( A ) HEK cells were transfected with vector (vec) control or FLAG-tagged Iso1 (1FL) or Iso2 (2FL). Lysates and supernatants were subject to a plate-based binding assay with CD30 coated on the plates and anti-FLAG antibody was used for detection. ( B ) Untagged Iso1 (1NT) was transfected into HEK cells with or without FLAG-tagged Iso2 (2FL), as well as 2FL alone. Cell lysates were subject to a plate-based binding assay. An anti-Iso1-specific CD30L antibody was used for capture and an anti-FLAG antibody for detection. ( C ) HEK cells were transfected with increasing amounts of 1NT plasmid, with either vector (vec, undetectable) control or 2FL at a constant amount and lysates were subject to a plate-based binding assay with recombinant CD30 coated on the plates. Anti-FLAG antibody was used for the detection of 2FL. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Clones were tested and chosen for response to CD30L-Fc recombinant protein (Sino Biological 10040-H01H, Wayne, PA, USA) via luciferase assay (described below).

Techniques: Transfection, Plasmid Preparation, Binding Assay, Recombinant

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso1 induces cytokine production in co-culture with CD30+ cells while Iso2 has no effect. Increasing quantities of HEK cells were transfected with either vector (vec) or CD30L isoforms with (FL) or without (NT) FLAG tag. After transfection, K299 cells were co-cultured with HEK cells for 24 h. Supernatants were harvested and subject to cytokine analysis by MSD assay. Error bars for all are from standard deviations between technical replicates.

Article Snippet: Clones were tested and chosen for response to CD30L-Fc recombinant protein (Sino Biological 10040-H01H, Wayne, PA, USA) via luciferase assay (described below).

Techniques: Co-Culture Assay, Transfection, Plasmid Preparation, FLAG-tag, Cell Culture

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 partially blocks Iso1-mediated stimulation of K299 cells. ( A ) HEK cells were transfected with either vector (vec) control or untagged Iso1 (1NT) with or without untagged Iso2 (2NT) or FLAG-tagged Iso2 (2FL). Transfected HEK cells were co-cultured with a K299-NFkB-Luc reporter clonal line. Two asterisks indicate p ≤ 0.01, three indicate p ≤ 0.001, and four indicate p ≤ 0.0001. ( B , C ) HEK cells were transfected with increasing quantities of 1NT plasmid with either vec or 2NT at a constant quantity, and then co-cultured with the K299-NFkB-Luc reporter line. Reporter activity ( B ) and IL-6 production ( C ) were measured. ( D ) HEK cells were transfected with either 1NT and vec or Iso2 and assessed for 1NT expression by flow cytometry. ( E ) Cells were transfected with increasing quantities of 1NT and either vec or 2FL, and the lysates were subject to SDS-PAGE and Western blot using a pan-CD30L antibody. Densitometry was used to quantify Iso1 bands, normalizing to GAPDH. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Clones were tested and chosen for response to CD30L-Fc recombinant protein (Sino Biological 10040-H01H, Wayne, PA, USA) via luciferase assay (described below).

Techniques: Transfection, Plasmid Preparation, Cell Culture, Activity Assay, Expressing, Flow Cytometry, SDS Page, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 can block Iso1/CD30 interaction and anti-Iso1 antibody binding. ( A ) HEK cells were transfected with increasing quantities of untagged Iso1 (1NT) plasmid with a constant amount of either vector (vec) control or FLAG-tagged Iso2 (2FL). Lysates were used in a plate-based binding assay using CD30 recombinant protein as the capture and an Iso1-specific anti-CD30L antibody was used for detection. ( B ) HEK cells were transfected with 1NT with or without 2FL, and 2FL alone. The lysates were used in a plate-based binding assay using an anti-pan-CD30L antibody for capture and an Iso1-specific anti-CD30L antibody for detection. ( C , D ) HEK cells were transfected with two quantities of 1NT and a constant quantity of 2FL or vector (vec) control in 10 cm plates. Additionally, 10% of the lysates were set aside as the input whole-cell lysate fraction ( C ), and the remainder was incubated with CD30-Fc recombinant protein and protein A/G beads ( D ). Both were subject to SDS-PAGE and Western blot. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Clones were tested and chosen for response to CD30L-Fc recombinant protein (Sino Biological 10040-H01H, Wayne, PA, USA) via luciferase assay (described below).

Techniques: Blocking Assay, Binding Assay, Transfection, Plasmid Preparation, Recombinant, Incubation, SDS Page, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: The CD30 ligand isoforms have distinct C-terminal extracellular regions. ( A ) Protein alignment of the two isoforms using Clustal Omega. Red—shared intracellular domain; cyan—shared transmembrane domain; purple—shared extracellular domain; black—predicted alpha helix within shared extracellular domain; yellow—extracellular sequences unique to each isoform. Asterisks indicate matching amino acid identity, dots and double dots indicate similarity, and hyphen indicates absence of residues in Iso2. The N-terminus for both proteins is intracellular and identical through residue 136, which includes the transmembrane domain at residues 38–62. The residues following 136 are distinct extracellular C-termini. ( B ) The predicted structural homology of CD30L isoforms to known protein structures generated by the Phyre2 web portal. Iso1 ( left ) showed strong homology to human TNF-alpha with several antiparallel beta-sheets. However, the software did not predict the presence of strong secondary structures for Iso2 ( right ), instead matching homology to small portions of proteins with unstructured domains. The two isoforms did share a predicted alpha helix that was membrane proximal on the extracellular side, present on the left side of each image.

Article Snippet: Coating antibodies included Iso1-specific anti-CD30L (R&D MAB1028, Minneapolis, MN, USA), pan-anti-CD30L (LSBio C293348, Shirley, MA, USA), and anti-FLAG (Sigma-Aldrich F3165, Saint Louis, MO, USA).

Techniques: Residue, Generated, Software, Membrane

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Both CD30L isoforms were expressed at the RNA level in primary human cells. ( A ) PBMCs were isolated from healthy donors ( n = 6). Cells were either untreated (nt) or stimulated with DMSO, PMA/ionomycin (P/I), lipopolysaccharide (LPS) isolated from E . coli 0111:B4 (LPS), anti-CD3/CD28 beads (CD3/28), or immune complex (IC) for 6 or 24 h. The graphs show actin-normalized expression of both isoforms at both timepoints for all stimulation conditions. Single asterisks indicate p ≤ 0.05 and double asterisks indicate p ≤ 0.01 between the conditions indicated. Error bars represent standard deviation between donors. ( B ) PBMCs were isolated from healthy donors ( n = 3, half of the 6 total donors). Cells were either untreated (nt) or stimulated with PMA/ionomycin (P/I) or immune complex (IC) for 24 h. After incubation, cells were subject to flow cytometry for B cell marker CD19, monocyte marker CD14, and T cell marker CD3, as well as CD30 ligand Iso1. PBMC cell types were present at expected ratios. Error bars are standard deviation between donors.

Article Snippet: Coating antibodies included Iso1-specific anti-CD30L (R&D MAB1028, Minneapolis, MN, USA), pan-anti-CD30L (LSBio C293348, Shirley, MA, USA), and anti-FLAG (Sigma-Aldrich F3165, Saint Louis, MO, USA).

Techniques: Isolation, Expressing, Standard Deviation, Incubation, Flow Cytometry, Marker

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 has a significantly impaired ability to bind CD30 compared to Iso1, but both are present in an exogenous CD30 protein complex. ( A ) HEK cells were transfected with vector (vec) control or FLAG-tagged Iso1 (1FL) or Iso2 (2FL). Lysates and supernatants were subject to a plate-based binding assay with CD30 coated on the plates and anti-FLAG antibody was used for detection. ( B ) Untagged Iso1 (1NT) was transfected into HEK cells with or without FLAG-tagged Iso2 (2FL), as well as 2FL alone. Cell lysates were subject to a plate-based binding assay. An anti-Iso1-specific CD30L antibody was used for capture and an anti-FLAG antibody for detection. ( C ) HEK cells were transfected with increasing amounts of 1NT plasmid, with either vector (vec, undetectable) control or 2FL at a constant amount and lysates were subject to a plate-based binding assay with recombinant CD30 coated on the plates. Anti-FLAG antibody was used for the detection of 2FL. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Coating antibodies included Iso1-specific anti-CD30L (R&D MAB1028, Minneapolis, MN, USA), pan-anti-CD30L (LSBio C293348, Shirley, MA, USA), and anti-FLAG (Sigma-Aldrich F3165, Saint Louis, MO, USA).

Techniques: Transfection, Plasmid Preparation, Binding Assay, Recombinant

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso1 induces cytokine production in co-culture with CD30+ cells while Iso2 has no effect. Increasing quantities of HEK cells were transfected with either vector (vec) or CD30L isoforms with (FL) or without (NT) FLAG tag. After transfection, K299 cells were co-cultured with HEK cells for 24 h. Supernatants were harvested and subject to cytokine analysis by MSD assay. Error bars for all are from standard deviations between technical replicates.

Article Snippet: Coating antibodies included Iso1-specific anti-CD30L (R&D MAB1028, Minneapolis, MN, USA), pan-anti-CD30L (LSBio C293348, Shirley, MA, USA), and anti-FLAG (Sigma-Aldrich F3165, Saint Louis, MO, USA).

Techniques: Co-Culture Assay, Transfection, Plasmid Preparation, FLAG-tag, Cell Culture

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 partially blocks Iso1-mediated stimulation of K299 cells. ( A ) HEK cells were transfected with either vector (vec) control or untagged Iso1 (1NT) with or without untagged Iso2 (2NT) or FLAG-tagged Iso2 (2FL). Transfected HEK cells were co-cultured with a K299-NFkB-Luc reporter clonal line. Two asterisks indicate p ≤ 0.01, three indicate p ≤ 0.001, and four indicate p ≤ 0.0001. ( B , C ) HEK cells were transfected with increasing quantities of 1NT plasmid with either vec or 2NT at a constant quantity, and then co-cultured with the K299-NFkB-Luc reporter line. Reporter activity ( B ) and IL-6 production ( C ) were measured. ( D ) HEK cells were transfected with either 1NT and vec or Iso2 and assessed for 1NT expression by flow cytometry. ( E ) Cells were transfected with increasing quantities of 1NT and either vec or 2FL, and the lysates were subject to SDS-PAGE and Western blot using a pan-CD30L antibody. Densitometry was used to quantify Iso1 bands, normalizing to GAPDH. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Coating antibodies included Iso1-specific anti-CD30L (R&D MAB1028, Minneapolis, MN, USA), pan-anti-CD30L (LSBio C293348, Shirley, MA, USA), and anti-FLAG (Sigma-Aldrich F3165, Saint Louis, MO, USA).

Techniques: Transfection, Plasmid Preparation, Cell Culture, Activity Assay, Expressing, Flow Cytometry, SDS Page, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 can block Iso1/CD30 interaction and anti-Iso1 antibody binding. ( A ) HEK cells were transfected with increasing quantities of untagged Iso1 (1NT) plasmid with a constant amount of either vector (vec) control or FLAG-tagged Iso2 (2FL). Lysates were used in a plate-based binding assay using CD30 recombinant protein as the capture and an Iso1-specific anti-CD30L antibody was used for detection. ( B ) HEK cells were transfected with 1NT with or without 2FL, and 2FL alone. The lysates were used in a plate-based binding assay using an anti-pan-CD30L antibody for capture and an Iso1-specific anti-CD30L antibody for detection. ( C , D ) HEK cells were transfected with two quantities of 1NT and a constant quantity of 2FL or vector (vec) control in 10 cm plates. Additionally, 10% of the lysates were set aside as the input whole-cell lysate fraction ( C ), and the remainder was incubated with CD30-Fc recombinant protein and protein A/G beads ( D ). Both were subject to SDS-PAGE and Western blot. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Coating antibodies included Iso1-specific anti-CD30L (R&D MAB1028, Minneapolis, MN, USA), pan-anti-CD30L (LSBio C293348, Shirley, MA, USA), and anti-FLAG (Sigma-Aldrich F3165, Saint Louis, MO, USA).

Techniques: Blocking Assay, Binding Assay, Transfection, Plasmid Preparation, Recombinant, Incubation, SDS Page, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: The CD30 ligand isoforms have distinct C-terminal extracellular regions. ( A ) Protein alignment of the two isoforms using Clustal Omega. Red—shared intracellular domain; cyan—shared transmembrane domain; purple—shared extracellular domain; black—predicted alpha helix within shared extracellular domain; yellow—extracellular sequences unique to each isoform. Asterisks indicate matching amino acid identity, dots and double dots indicate similarity, and hyphen indicates absence of residues in Iso2. The N-terminus for both proteins is intracellular and identical through residue 136, which includes the transmembrane domain at residues 38–62. The residues following 136 are distinct extracellular C-termini. ( B ) The predicted structural homology of CD30L isoforms to known protein structures generated by the Phyre2 web portal. Iso1 ( left ) showed strong homology to human TNF-alpha with several antiparallel beta-sheets. However, the software did not predict the presence of strong secondary structures for Iso2 ( right ), instead matching homology to small portions of proteins with unstructured domains. The two isoforms did share a predicted alpha helix that was membrane proximal on the extracellular side, present on the left side of each image.

Article Snippet: Recombinant Iso1 CD30L-Fc protein (Sino Biological 10040-H01H, Wayne, PA, USA) and transfected HEK cell culture were used to stimulate CD30+ cells.

Techniques: Residue, Generated, Software, Membrane

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Both CD30L isoforms were expressed at the RNA level in primary human cells. ( A ) PBMCs were isolated from healthy donors ( n = 6). Cells were either untreated (nt) or stimulated with DMSO, PMA/ionomycin (P/I), lipopolysaccharide (LPS) isolated from E . coli 0111:B4 (LPS), anti-CD3/CD28 beads (CD3/28), or immune complex (IC) for 6 or 24 h. The graphs show actin-normalized expression of both isoforms at both timepoints for all stimulation conditions. Single asterisks indicate p ≤ 0.05 and double asterisks indicate p ≤ 0.01 between the conditions indicated. Error bars represent standard deviation between donors. ( B ) PBMCs were isolated from healthy donors ( n = 3, half of the 6 total donors). Cells were either untreated (nt) or stimulated with PMA/ionomycin (P/I) or immune complex (IC) for 24 h. After incubation, cells were subject to flow cytometry for B cell marker CD19, monocyte marker CD14, and T cell marker CD3, as well as CD30 ligand Iso1. PBMC cell types were present at expected ratios. Error bars are standard deviation between donors.

Article Snippet: Recombinant Iso1 CD30L-Fc protein (Sino Biological 10040-H01H, Wayne, PA, USA) and transfected HEK cell culture were used to stimulate CD30+ cells.

Techniques: Isolation, Expressing, Standard Deviation, Incubation, Flow Cytometry, Marker

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 has a significantly impaired ability to bind CD30 compared to Iso1, but both are present in an exogenous CD30 protein complex. ( A ) HEK cells were transfected with vector (vec) control or FLAG-tagged Iso1 (1FL) or Iso2 (2FL). Lysates and supernatants were subject to a plate-based binding assay with CD30 coated on the plates and anti-FLAG antibody was used for detection. ( B ) Untagged Iso1 (1NT) was transfected into HEK cells with or without FLAG-tagged Iso2 (2FL), as well as 2FL alone. Cell lysates were subject to a plate-based binding assay. An anti-Iso1-specific CD30L antibody was used for capture and an anti-FLAG antibody for detection. ( C ) HEK cells were transfected with increasing amounts of 1NT plasmid, with either vector (vec, undetectable) control or 2FL at a constant amount and lysates were subject to a plate-based binding assay with recombinant CD30 coated on the plates. Anti-FLAG antibody was used for the detection of 2FL. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Recombinant Iso1 CD30L-Fc protein (Sino Biological 10040-H01H, Wayne, PA, USA) and transfected HEK cell culture were used to stimulate CD30+ cells.

Techniques: Transfection, Plasmid Preparation, Binding Assay, Recombinant

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso1 induces cytokine production in co-culture with CD30+ cells while Iso2 has no effect. Increasing quantities of HEK cells were transfected with either vector (vec) or CD30L isoforms with (FL) or without (NT) FLAG tag. After transfection, K299 cells were co-cultured with HEK cells for 24 h. Supernatants were harvested and subject to cytokine analysis by MSD assay. Error bars for all are from standard deviations between technical replicates.

Article Snippet: Recombinant Iso1 CD30L-Fc protein (Sino Biological 10040-H01H, Wayne, PA, USA) and transfected HEK cell culture were used to stimulate CD30+ cells.

Techniques: Co-Culture Assay, Transfection, Plasmid Preparation, FLAG-tag, Cell Culture

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 partially blocks Iso1-mediated stimulation of K299 cells. ( A ) HEK cells were transfected with either vector (vec) control or untagged Iso1 (1NT) with or without untagged Iso2 (2NT) or FLAG-tagged Iso2 (2FL). Transfected HEK cells were co-cultured with a K299-NFkB-Luc reporter clonal line. Two asterisks indicate p ≤ 0.01, three indicate p ≤ 0.001, and four indicate p ≤ 0.0001. ( B , C ) HEK cells were transfected with increasing quantities of 1NT plasmid with either vec or 2NT at a constant quantity, and then co-cultured with the K299-NFkB-Luc reporter line. Reporter activity ( B ) and IL-6 production ( C ) were measured. ( D ) HEK cells were transfected with either 1NT and vec or Iso2 and assessed for 1NT expression by flow cytometry. ( E ) Cells were transfected with increasing quantities of 1NT and either vec or 2FL, and the lysates were subject to SDS-PAGE and Western blot using a pan-CD30L antibody. Densitometry was used to quantify Iso1 bands, normalizing to GAPDH. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Recombinant Iso1 CD30L-Fc protein (Sino Biological 10040-H01H, Wayne, PA, USA) and transfected HEK cell culture were used to stimulate CD30+ cells.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Activity Assay, Expressing, Flow Cytometry, SDS Page, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 can block Iso1/CD30 interaction and anti-Iso1 antibody binding. ( A ) HEK cells were transfected with increasing quantities of untagged Iso1 (1NT) plasmid with a constant amount of either vector (vec) control or FLAG-tagged Iso2 (2FL). Lysates were used in a plate-based binding assay using CD30 recombinant protein as the capture and an Iso1-specific anti-CD30L antibody was used for detection. ( B ) HEK cells were transfected with 1NT with or without 2FL, and 2FL alone. The lysates were used in a plate-based binding assay using an anti-pan-CD30L antibody for capture and an Iso1-specific anti-CD30L antibody for detection. ( C , D ) HEK cells were transfected with two quantities of 1NT and a constant quantity of 2FL or vector (vec) control in 10 cm plates. Additionally, 10% of the lysates were set aside as the input whole-cell lysate fraction ( C ), and the remainder was incubated with CD30-Fc recombinant protein and protein A/G beads ( D ). Both were subject to SDS-PAGE and Western blot. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Recombinant Iso1 CD30L-Fc protein (Sino Biological 10040-H01H, Wayne, PA, USA) and transfected HEK cell culture were used to stimulate CD30+ cells.

Techniques: Blocking Assay, Binding Assay, Transfection, Plasmid Preparation, Recombinant, Incubation, SDS Page, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: The CD30 ligand isoforms have distinct C-terminal extracellular regions. ( A ) Protein alignment of the two isoforms using Clustal Omega. Red—shared intracellular domain; cyan—shared transmembrane domain; purple—shared extracellular domain; black—predicted alpha helix within shared extracellular domain; yellow—extracellular sequences unique to each isoform. Asterisks indicate matching amino acid identity, dots and double dots indicate similarity, and hyphen indicates absence of residues in Iso2. The N-terminus for both proteins is intracellular and identical through residue 136, which includes the transmembrane domain at residues 38–62. The residues following 136 are distinct extracellular C-termini. ( B ) The predicted structural homology of CD30L isoforms to known protein structures generated by the Phyre2 web portal. Iso1 ( left ) showed strong homology to human TNF-alpha with several antiparallel beta-sheets. However, the software did not predict the presence of strong secondary structures for Iso2 ( right ), instead matching homology to small portions of proteins with unstructured domains. The two isoforms did share a predicted alpha helix that was membrane proximal on the extracellular side, present on the left side of each image.

Article Snippet: Open reading frames (ORFs) for both the CD30L isoforms were purchased from Origene (RC211276, RC231923, Rockville, MD, USA), containing C-terminal FLAG/MYC epitope tags.

Techniques: Residue, Generated, Software, Membrane

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Both CD30L isoforms were expressed at the RNA level in primary human cells. ( A ) PBMCs were isolated from healthy donors ( n = 6). Cells were either untreated (nt) or stimulated with DMSO, PMA/ionomycin (P/I), lipopolysaccharide (LPS) isolated from E . coli 0111:B4 (LPS), anti-CD3/CD28 beads (CD3/28), or immune complex (IC) for 6 or 24 h. The graphs show actin-normalized expression of both isoforms at both timepoints for all stimulation conditions. Single asterisks indicate p ≤ 0.05 and double asterisks indicate p ≤ 0.01 between the conditions indicated. Error bars represent standard deviation between donors. ( B ) PBMCs were isolated from healthy donors ( n = 3, half of the 6 total donors). Cells were either untreated (nt) or stimulated with PMA/ionomycin (P/I) or immune complex (IC) for 24 h. After incubation, cells were subject to flow cytometry for B cell marker CD19, monocyte marker CD14, and T cell marker CD3, as well as CD30 ligand Iso1. PBMC cell types were present at expected ratios. Error bars are standard deviation between donors.

Article Snippet: Open reading frames (ORFs) for both the CD30L isoforms were purchased from Origene (RC211276, RC231923, Rockville, MD, USA), containing C-terminal FLAG/MYC epitope tags.

Techniques: Isolation, Expressing, Standard Deviation, Incubation, Flow Cytometry, Marker

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 has a significantly impaired ability to bind CD30 compared to Iso1, but both are present in an exogenous CD30 protein complex. ( A ) HEK cells were transfected with vector (vec) control or FLAG-tagged Iso1 (1FL) or Iso2 (2FL). Lysates and supernatants were subject to a plate-based binding assay with CD30 coated on the plates and anti-FLAG antibody was used for detection. ( B ) Untagged Iso1 (1NT) was transfected into HEK cells with or without FLAG-tagged Iso2 (2FL), as well as 2FL alone. Cell lysates were subject to a plate-based binding assay. An anti-Iso1-specific CD30L antibody was used for capture and an anti-FLAG antibody for detection. ( C ) HEK cells were transfected with increasing amounts of 1NT plasmid, with either vector (vec, undetectable) control or 2FL at a constant amount and lysates were subject to a plate-based binding assay with recombinant CD30 coated on the plates. Anti-FLAG antibody was used for the detection of 2FL. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Open reading frames (ORFs) for both the CD30L isoforms were purchased from Origene (RC211276, RC231923, Rockville, MD, USA), containing C-terminal FLAG/MYC epitope tags.

Techniques: Transfection, Plasmid Preparation, Control, Binding Assay, Recombinant

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso1 induces cytokine production in co-culture with CD30+ cells while Iso2 has no effect. Increasing quantities of HEK cells were transfected with either vector (vec) or CD30L isoforms with (FL) or without (NT) FLAG tag. After transfection, K299 cells were co-cultured with HEK cells for 24 h. Supernatants were harvested and subject to cytokine analysis by MSD assay. Error bars for all are from standard deviations between technical replicates.

Article Snippet: Open reading frames (ORFs) for both the CD30L isoforms were purchased from Origene (RC211276, RC231923, Rockville, MD, USA), containing C-terminal FLAG/MYC epitope tags.

Techniques: Co-Culture Assay, Transfection, Plasmid Preparation, FLAG-tag, Cell Culture

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 partially blocks Iso1-mediated stimulation of K299 cells. ( A ) HEK cells were transfected with either vector (vec) control or untagged Iso1 (1NT) with or without untagged Iso2 (2NT) or FLAG-tagged Iso2 (2FL). Transfected HEK cells were co-cultured with a K299-NFkB-Luc reporter clonal line. Two asterisks indicate p ≤ 0.01, three indicate p ≤ 0.001, and four indicate p ≤ 0.0001. ( B , C ) HEK cells were transfected with increasing quantities of 1NT plasmid with either vec or 2NT at a constant quantity, and then co-cultured with the K299-NFkB-Luc reporter line. Reporter activity ( B ) and IL-6 production ( C ) were measured. ( D ) HEK cells were transfected with either 1NT and vec or Iso2 and assessed for 1NT expression by flow cytometry. ( E ) Cells were transfected with increasing quantities of 1NT and either vec or 2FL, and the lysates were subject to SDS-PAGE and Western blot using a pan-CD30L antibody. Densitometry was used to quantify Iso1 bands, normalizing to GAPDH. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Open reading frames (ORFs) for both the CD30L isoforms were purchased from Origene (RC211276, RC231923, Rockville, MD, USA), containing C-terminal FLAG/MYC epitope tags.

Techniques: Transfection, Plasmid Preparation, Control, Cell Culture, Activity Assay, Expressing, Flow Cytometry, SDS Page, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: The Opposite Functions of CD30 Ligand Isoforms

doi: 10.3390/cimb46030172

Figure Lengend Snippet: Iso2 can block Iso1/CD30 interaction and anti-Iso1 antibody binding. ( A ) HEK cells were transfected with increasing quantities of untagged Iso1 (1NT) plasmid with a constant amount of either vector (vec) control or FLAG-tagged Iso2 (2FL). Lysates were used in a plate-based binding assay using CD30 recombinant protein as the capture and an Iso1-specific anti-CD30L antibody was used for detection. ( B ) HEK cells were transfected with 1NT with or without 2FL, and 2FL alone. The lysates were used in a plate-based binding assay using an anti-pan-CD30L antibody for capture and an Iso1-specific anti-CD30L antibody for detection. ( C , D ) HEK cells were transfected with two quantities of 1NT and a constant quantity of 2FL or vector (vec) control in 10 cm plates. Additionally, 10% of the lysates were set aside as the input whole-cell lysate fraction ( C ), and the remainder was incubated with CD30-Fc recombinant protein and protein A/G beads ( D ). Both were subject to SDS-PAGE and Western blot. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown.

Article Snippet: Open reading frames (ORFs) for both the CD30L isoforms were purchased from Origene (RC211276, RC231923, Rockville, MD, USA), containing C-terminal FLAG/MYC epitope tags.

Techniques: Blocking Assay, Binding Assay, Transfection, Plasmid Preparation, Control, Recombinant, Incubation, SDS Page, Western Blot