(A) Nonstarved and starved N2 cells were fixed and analyzed by immunofluorescence microscopy with an anti-MUC5AC antibody (green). The nuclear DNA was stained with DAPI (blue) to localize the …
(A) Illustration of the mucin secretion assay. Starved N2 cells are treated with PMA. Secreted MUC5AC is fixed on the cell surface and labeled with anti-MUC5AC antibodies followed by quantitative …
Starved N2 cells were treated for the indicated times with 2 μM PMA, fixed with formaldehyde and the amount of secreted MUC5AC bound to the cell surface was detected with anti-MUC5AC antibody and …
(A) Screening procedure. N2 cells were serum starved for 6 days. The cells were then seeded into the wells of 96-well plates and transfected in triplicates on three sets of plates with siRNAs …
N2 cells were starved for 6 days and transfected with the corresponding siRNA on day 6. The cells were grown for 3 additional days, pulsed with 35S-methionine and chased with cold …
(A) Total RNA was extracted from nonstarved- and 5-day starved N2 cells. 1 μg of total RNA was used to generate cDNA by reverse transcription. PCR was performed on cDNA with primers specific for the …
(A) Total RNA was extracted from control and TRPM5 stable knockdown (TRPM5 KD) cells and analyzed for knockdown efficiency of TRPM5 on mRNA level by quantitative real-time PCR using primers specific …
(A) Nonstarved and starved N2 cells stably transfected with HA-TRPM5 were fixed and analyzed by immunofluorescence microscopy with an anti-HA antibody (green) and an anti-MUC5AC antibody (red). The …
(A) Control and TRPM5 stable knockdown cells were starved and incubated for 30 min at 37°C with 100 μM ATP. Secreted MUC5AC was collected and processed for dot blot analysis with an anti-MUC5AC …
Starved N2 cells were treated for 30 min with the indicated concentrations of thapsigargin and ionomycin. Secreted MUC5AC was collected and processed for dot blot analysis with an anti-MUC5AC …
(A) Time course of mean Ca2+ responses (Fura-2 ratio) obtained in starved N2 cells treated with 100 μM ATP in the presence (n = 138) or absence of 1.2 mM Ca2+ (n = 118) in the extracellular …
(A) Starved N2 cells were preincubated for 15 min with or without KB-R7943 (50 μM) followed by incubation with 100 μM ATP in the presence or absence of KB-R7943. Secreted MUC5AC was analyzed by dot …
(A) Current traces (bottom) obtained in a starved N2 cell held at −80 mV and pulsed from −60 mV to +60 mV in 5 mV voltage steps (top). Traces are representative of six cells recorded under identical …
Thirty-four proteins involved in MUC5AC secretion
Gene ID | Symbol | MUC5AC secretion | Type | Known localization | Expression in N2 cells |
---|---|---|---|---|---|
6833 | SUR1 | Hyposecretory | Transporter | PM | Detected |
2900 | GRIK4 | Hyposecretory | Ion channel | PM | Detected |
29,850 | TRPM5 | Hyposecretory | Ion channel | PM | Detected |
6326 | SCN2A | Hyposecretory | Ion channel | PM | Not detected |
6328 | SCN3A | Hyposecretory | Ion channel | PM | Not detected |
6915 | TBXA2R | Hyposecretory | GPCR | PM | Not detected |
6753 | SSTR3 | Hyposecretory | GPCR | PM | Not detected |
122042 | RXFP2 | Hyposecretory | GPCR | PM | Not detected |
1394 | CRHR1 | Hyposecretory | GPCR | PM | Not detected |
10,803 | CCR9 | Hyposecretory | GPCR | PM | Detected |
524 | CX3CR1 | Hyposecretory | GPCR | PM | Not detected |
8484 | GALR3 | Hyposecretory | GPCR | PM | Not detected |
83,550 | GPR101 | Hyposecretory | GPCR | PM | Not detected |
118442 | GPR62 | Hyposecretory | GPCR | PM | Detected |
4914 | NTRK1 | Hyposecretory | TM receptor | PM | Not detected |
9437 | NCR1 | Hyposecretory | TM receptor | PM | Not detected |
55,824 | PAG1 | Hyposecretory | Other | PM | Detected |
259215 | LY6G6F | Hyposecretory | TM receptor | PM | Not detected |
56,140 | PCDHA8 | Hyposecretory | Cadherin | PM | Not detected |
225689 | MAPK15 | Hyposecretory | Kinase | Cytosol | Detected |
23,542 | MAPK8IP2 | Hyposecretory | Other | Cytosol | Not detected |
10,454 | TAB1 | Hyposecretory | Scaffold | Cytosol | Detected |
6490 | SILV | Hyposecretory | Melanosome biogenesis | Melanosome | Detected |
6494 | SIPA1 | Hyposecretory | GTPase activator | Cytosol, nucleus | Detected |
30,818 | KCNIP3 | Hyposecretory | Ca2+ binding | Palmitoylated: membrane, cytosol | Detected |
22,926 | ATF6 | Hyposecretory | Transcription | ER | Detected |
26,499 | PLEK2 | Hyposecretory | Actin binding | Cytoskeleton | Detected |
4790 | NFKB1 | Hyposecretory | Transcription | Nucleus, cytosol | Detected |
6720 | SREBF1 | Hyposecretory | Transcription | ER | Detected |
1238 | CCBP2 | Hypersecretory | GPCR | PM | Detected |
84,033 | OBSCN | Hypersecretory | Kinase | Cytosol | NT |
9373 | PLAA | Hypersecretory | Other | Cytosol | NT |
4843 | NOS2 | Hypersecretory | Other | Cytosol | NT |
4893 | NRAS | Hypersecretory | G-protein | Cytosol | NT |
ER: endoplasmic reticulum; GPCR: G protein coupled receptor; PM: plasma membrane; TM receptor: transmembrane receptor; NT: not tested.
Identification of proteins involved in MUC5AC secretion (PIMS)
Gene | PIMS | MUC5AC secretion | Type | Known localization | Expression in starved N2 cells | Disease |
---|---|---|---|---|---|---|
SUR1 | 1 | Hyposecretory | Transporter | PM | Unchanged | Diabetes mellitus, insulin secretion (Aittoniemi et al., 2009) |
GRIK4 | 2 | Hyposecretory | Ion channel | PM | Upregulated | |
TRPM5 | 3 | Hyposecretory | Ion channel | PM | Unchanged | Insulin secretion (Colsoul et al., 2010) |
KCNIP3 | 4 | Hyposecretory | Ca2+ binding | Palmitoylated membrane, cytosol | Upregulated | |
SIPA1 | 5 | Hyposecretory | GTPase activator | Cytosol, nucleus | Unchanged | |
PLEK2 | 6 | Hyposecretory | Actin binding | Cytoskeleton | Unchanged | |
TAB1 | 7 | Hyposecretory | Scaffold | Cytosol | Unchanged | |
MAPK15 | 8 | Hyposecretory | Kinase | Cytosol | Upregulated | |
SILV | 9 | Hyposecretory | Melanosome biogenesis | Melanosome | Unchanged | |
SREBF1 | 10 | Hyposecretory | Transcription regulator | ER | Unchanged | |
ATF6 | 11 | Hyposecretory | Transcription regulator | ER | Unchanged | |
NFKB1 | 12 | Hyposecretory | Transcription regulator | Cytosol, nucleus | Unchanged | MUC5AC biosynthesis (Fujisawa et al., 2009); asthma (Hart et al., 1998); COPD (di Stefano et al., 2002) |
PAG1 | 13 | Hyposecretory | Other | PM | Unchanged | |
CCR9 | 14 | Hyposecretory | GPCR | PM | Upregulated | Inflammatory bowel disease (Nishimura et al., 2009) |
GPR62 | 15 | Hyposecretory | GPCR | PM | Unchanged | |
CCBP2 | 16 | Hypersecretory | GPCR | PM | Unchanged |
ER: endoplasmic reticulum; GPCR: G protein-coupled receptor; PM: plasma membrane; TM receptor: transmembrane receptor.
Primer sequences used for detecting mRNA for the respective PIMS in N2 cells
Gene | Forward primer 5′–3′ | Reverse primer 5′–3′ |
---|---|---|
TRPM5 | GTGGCCATCTTCCTGTTCAT | CTTCATCATGCGCTCTACCA |
CCR9 | GCCAGCCTTGGCCCTGTTGT | TCCAGCAAGGCCTGCGCTTC |
PLEK2 | AGAACAGGCCAGTGGGTGGGT | GCTCGCTCAGCCTTGCTGCT |
TAB1 | TCAATCATCGCAGCAATCTC | GGCTACACGGACATTGACCT |
KCNIP3 | CCACCACCTATGCACACTTCC | GTCGTAGAGATTAAAGGCCCAC |
SILV | GGGCTACAAAAGTACCCAGAAAC | CCTTGAGGGACACTTGACCAC |
SIPA1 | CTCCTTTCTGCCACGTACCTT | TTTTGGAGTTCCCTTAGGGTCT |
HPRT1 | TGACACTGGCAAAACAATGCA | GGTCCTTTTCACCAGCAAGCT |
MUC5AC | CAACCCCTCCTACTGCTACG | CTGGTGCTGAAGAGGGTCAT |
GPR62 | GGTGGTTTCCGTGGGGGCTC | TGGGCCCAGACCGCAGGATT |
PAG1 | TGGACGGCAGCCATGCATCC | ACTGTTGGTGTGGGCAGCGG |
ATF6 | AGGTGGGTAGCGGTTGGGAGG | GCGGCACCTTACAGGCACCC |
SREBF1 | CCACGGCAGCCCCTGTAACG | GGGACTGAGACCTGCCGCCT |
MAPK15 | TACAACAGGTCCCTCCCCGGC | CCCAGTGCCGAGTGGCAGAC |
SUR1 | GCCTTCGCAGACCGCACTGT | CTGCACGGACGAAGGAGGCG |
NFKB1 | CGCCACCCGGCTTCAGAATGG | GTATGGGCCATCTGCTGTTGGCA |
CCBP2 | CGGCGGGCATGGGACCATTT | AAGGCCACCACCAAGGCTGC |
GRIK4 | CGTGGCTCGTGATGGTCGCC | GCCTCTCAGGAGCGCGGTTG |
GAPDH | TGCACCACCAACTGCTTAGC | GGCATGGACTGTGGTCATGAG |
List of positive hits from primary screen.
Lists positive hits from the primary screen, their reason for exclusion, the B-score, the ranking product and the E-value.