Journal: Science (New York, N.Y.)

Article Title: A protein network map of head and neck cancer reveals PIK3CA mutant drug sensitivity

doi: 10.1126/science.abf2911

Figure Lengend Snippet: Key Resources

Article Snippet: Gαi-DREADD (pcDNA3.1) Antibodies RSK1/2/3 antibody Cell Signaling Technology 9355 ERK1/2 Cell Signaling Technology 4695 phospho-PAK1(S199/204)/PAK2(S192/197) Cell Signaling Technology 2605 PAK1 Cell Signaling Technology 2602 PAK2 Cell Signaling Technology 2608 pERK Cell Signaling Technology 9106 FGFR3 OriGene TA801078 Daple Millipore EMD ABS515 GAPDH Cell Signaling Technology 2118 secondary goat anti-rabbit HRP Southern Biotech 4010-05 P-HER3-Y1197 Cell Signaling Technology 4561 HER3 Cell Signaling Technology 12708 goat anti-mouse HRP Southern Biotech 1010-05 anti-B-tubulin Abcam ab6276 ERK Cell Signaling Technology 9102 Deposited data Unprocessed peptide files This paper PRIDE ProteomeXchange: PXD019469 Raw data This paper PRIDE ProteomeXchange: PXD019469 Chemicals, Peptides, and Recombinant Proteins Tris G-Biosciences RC108 Acetonitrile, HPLC grade (ACN) Thermo Fisher Scientific A955-4 cOmplete protease inhibitor cocktail tablets mini, EDTA-free Roche 11846 170 001 Dithiothreitol (DTT) Sigma-Aldrich 43819 Formic acid (FA) Thermo Fisher Scientific 28905 Iodoacetamide (IAA) Acros Organic 122270250 Sequencing-grade modified trypsin Promega V5111 Benzonase Sigma E1014-25KU Trifluoroacetic acid (TFA) Thermo Fisher Scientific 28904 Urea Sigma-Aldrich U5378-1kg Fetal bovine serum (FBS) Gibco A3160502 DMEM Corning MT10013CV DMEM/F12 Corning MT10092CV Water, HPLC grade Sigma-Aldrich 270733-4 L Igepal (NP-40) Sigma-Aldrich I3021 Minimal Essential Media Corning 10-009-CV Opti-MEM Thermo Fisher Scientific 31985062 BEGM™ (Lonza) Lonza CC-3170 1% Penicillin-Streptomycin Corning MT30002Cl Paraformaldehyde, 4% solution in PBS Thermo Scientific MFCD00133991 PolyJet SignaGen SL100688 Lipofectamine 3000 ThermoFisher Scientific L3000008 hydrocortisone Sigma H6909-10ML Rapigest Waters 186001861 3x Flag Peptide Sigma F4799-4MG Anti-Flag M2 Magnetic Beads Sigma M8823-5ML Lipofectamine RNAiMAX Thermo Fisher Scientific 100014472 siFGFR3 Sigma Aldrich SIHK0780, SIHK0781, SIHK0782 native coelenterazine Biotium 10110-1 pooled siControl Dharmacon D-001810-10-20 siDaple Dharmacon L-033364-01-0005 10μM clozapine-N-oxide Cayman Chemical NC1044836 5μM native coelenterazine Biotium 10110-1 RPS6KA1 siRNA pool OriGene SR304161 non-targeting control siRNA Dharmacon D-001810-10 Triton X-100 Thermo Scientific 9002-93-1 Software and Algorithms artMS Bioconductor https://www.bioconductor.org/packages/release/bioc/html/artMS.html MSstats Bioconductor https://bioconductor.org/packages/release/bioc/html/MSstats.html Skyline MacCoss Lab https://skyline.ms/project/home/begin.view?

Techniques: Mutagenesis, Recombinant, Protease Inhibitor, Sequencing, Modification, Magnetic Beads, Software, Mass Spectrometry

Journal: iScience

Article Title: The endoplasmic reticulum membrane complex promotes proteostasis of GABA A receptors

doi: 10.1016/j.isci.2022.104754

Figure Lengend Snippet: Effect of EMC3 and EMC6 on the protein levels and whole-cell patch-clamping currents of endogenous GABA A receptors (A) Mouse GT1-7 neurons were incubated with siRNA against EMC3 or EMC6 for 48 h. Proteins were extracted and analyzed by western blotting; normalized band intensity was shown below the images (n = 3), with β-actin as the loading control. (B) Mouse GT1-7 neurons were incubated with siRNA against EMC3 or EMC6 for 48 h. Whole-cell patch-clamping was performed on the cells with the IonFlux Mercury 16 ensemble plates at a holding potential of −60 mV. GABA (1 mM) was applied for 4 s, as indicated by the horizontal bar above the currents. The peak currents (Imax) were acquired and analyzed by Fluxion Data Analyzer (n = 6 - 10). NT: Non-targeting scrambled siRNA; pA, picoampere. (C) Confocal microscopy imaging of primary rat cortical neurons demonstrated reduced surface expression of GABA A receptors after siRNA treatment of EMC3 and EMC6 through lentivirus transduction. Lentiviruses were generated from transiently transfected HEK293T cells with the following plasmids and collected after 60 h from the media passing through 0.45 μm filter: EMC3- or EMC6-set of four siRNA lentivectors, packaging and envelop plasmids. At day-in-vitro (DIV) 6 of the primary rat cortical neurons, lentivirus transduction was carried out at a multiplicity-of-infection (MOI) of 10. At DIV 12, neurons were stained for cell surface GABA A receptor α1 subunits (top row), β2/β3 subunits (middle row), and γ2 subunits (bottom row), colored in red. DAPI staining for the nucleus was colored in blue. Scale bar = 20 μm. Quantification of the fluorescence intensity by using ImageJ was shown on the bottom after background correction from 20–30 neurons. Each data point is presented as mean ± SEM ∗, p< 0.05; ∗∗, p< 0.01.

Article Snippet: The human FLAG-tagged EMC5 plasmid (catalog #: RC207046) and FLAG-tagged EMC6 plasmid (catalog #: RC215548) were obtained from OriGene.

Techniques: Incubation, Western Blot, Confocal Microscopy, Imaging, Expressing, Transduction, Generated, Transfection, In Vitro, Infection, Staining, Fluorescence

Journal: iScience

Article Title: The endoplasmic reticulum membrane complex promotes proteostasis of GABA A receptors

doi: 10.1016/j.isci.2022.104754

Figure Lengend Snippet: EMC3 and EMC6 promote anterograde trafficking of GABA A receptors (A and B) Significant reduction of cell surface and total α1 and β2 subunits of GABA A receptors was observed when both EMC3 and EMC6 were knocked down. We carried out siRNA transfection in HEK293T cells stably expressing α1β2γ2 GABA A receptors. To test the surface expression of GABA A receptors, biotinylation experiments were performed 48 h after siRNA transfection of both EMC3 and EMC6 (A). Surface proteins were enriched through biotin-neutravidin affinity purification, and western blot analysis was applied to detect surface α1 and β2 subunits. Na + /K + ATPase served as loading control of cell surface proteins. To test the total protein expression of GABA A receptors, cells were lysed and total proteins were collected and subjected to SDS-PAGE and western blot analysis (B). β-actin was used as the total protein loading control. Normalized band intensity was shown on the right to the blots (n = 3). (C) The ratio of the surface/total subunits of GABA A receptors was quantified, as a measure of their surface trafficking efficiency. Data was taken from (A) and (B) for the calculation. (D) HEK293T cells stably expressing α1β2γ2 GABA A receptors were transfected with non-targeting siRNA or siRNAs against EMC3 and EMC6. 48 h after transfection, cycloheximide (CHX) (100 μg/mL), a potent protein synthesis inhibitor, was added to cell culture media for the indicated time. Cells were then harvested, and total proteins were subjected to SDS-PAGE and western blot analysis. Quantification of the α1 band intensity was plotted against the incubation time with CHX (n = 3). (E and F) EMC3 and EMC6 promote GABA A receptors’ trafficking from the ER to Golgi as demonstrated through endoglycosidase H (Endo H) digestion. EMC3 or EMC6 siRNA transfection was applied in HEK293T cells stably expressing α1β2γ2 GABA A receptors; 48 h after transfection, proteins were extracted, and subjected to Endo H digestion and western blot analysis. Endo H resistant bands (top two bands in lanes two and 4) represent proteins that have correctly folded in the ER, trafficked to Golgi and fully modified with the N -linked complex glycans, thus becoming resistant to Endo H. On the other hand, acting upon proteins remaining in ER, Endo H may remove the high mannose structure after the asparaginyl- N -acetyl-D-glucosamine on the α1 subunits, generating Endo H sensitive bands (bottom band in lanes two and 4). The Peptide-N-Glycosidase F (PNGase F) enzyme-treated samples served as a control for unglycosylated α1 subunits (lane 5). Quantification of the ratio of Endo H resistant/total α1 band intensity, as a measure of the trafficking efficiency of α1 subunits, was shown on the right (n = 3). (G) HEK293T cells stably expressing α1β2γ2 GABA A receptors were transfected with non-targeting siRNA or siRNAs against EMC3, EMC6, or both EMC3 and EMC6. 48 h after transfection, cells were harvested, and total proteins were subjected to SDS-PAGE and western blot analysis. Quantification of the normalized individual EMC subunit band intensity was shown on the right panels (n = 3). Each data point is presented as mean ± SEM ∗, p< 0.05; ∗∗, p< 0.01. NT: Non-targeting scrambled siRNA.

Article Snippet: The human FLAG-tagged EMC5 plasmid (catalog #: RC207046) and FLAG-tagged EMC6 plasmid (catalog #: RC215548) were obtained from OriGene.

Techniques: Transfection, Stable Transfection, Expressing, Affinity Purification, Western Blot, SDS Page, Cell Culture, Incubation, Modification

Journal: iScience

Article Title: The endoplasmic reticulum membrane complex promotes proteostasis of GABA A receptors

doi: 10.1016/j.isci.2022.104754

Figure Lengend Snippet: Interactions of EMC3 and EMC6 with neurotransmitter-gated ion channels (A) Co-immunoprecipitation (Co-IP) from primary rat cortical neurons demonstrated endogenous interactions between α1 subunits of GABA A receptors and EMC3, EMC6, and a number of α1-interacting chaperones (BiP and calnexin) and ERAD factors (Grp94 and VCP). Neurons were plated onto 10-cm dishes at a density of one million per dish. At DIV 12, proteins were extracted for Co-IP. IgG was used as a negative control during the immunoprecipitation. n = 3. (B) Co-IP from primary rat cortical neurons demonstrated endogenous interactions between EMC3/EMC6 and a number of ion channels, including N-methyl-D-aspartate receptors (NMDARs, including NR1, NR2A and NR2B subunits) and nicotinic acetylcholine receptors (nAChR α7 subunit). n = 3. (C) Co-IP from mouse cortical homogenates, which were prepared from C57BL/6J mice between 8 and 10 weeks of age, demonstrated endogenous interactions between EMC3/EMC6 and selected ion channels. n = 3. (D) Schematic of the primary sequence of EMC3 and EMC6. R31 and R180 in EMC3 and N22 and D27 in EMC6 were reported to influence the biogenesis of EMC-dependent client proteins. (E) Mutation of R31A or R180A in EMC3 significantly reduced the interaction of EMC3 with GABA A α1 subunits. The cDNAs of FLAG-tagged EMC3, either in the wild type (WT) form or carrying appropriate mutations of R31A or R180A, were transiently transfected in HEK293T cells stably expressing α1β2γ2 GABA A receptors. 48 h after transfection, proteins were extracted from cell lysates and incubated with anti-FLAG M2 magnetic beads. The immuno-purified eluents were separated through SDS-PAGE gel, and western blot analysis was performed to detect α1 subunits and FLAG. Quantification of the band intensity of α1 over FLAG after immunoprecipitation was shown on the right (n = 3). (F) Mutation of D27A or N22A in EMC6 significantly reduced the interaction of EMC6 with GABA A R α1 subunits. Transfection of cDNAs was applied similarly as in E, however with co-application of FLAG-tagged EMC5 and EMC6 variants in HEK293T cells stably expressing α1β2γ2 GABA A receptors. Co-IP and visualization of protein bands were carried out the same way as in E as well. Quantification of the band intensity of α1 over FLAG-tagged EMC6 after immunoprecipitation was shown on the right (n = 3). (G) Significant increase of the interaction of SEC61α and α1 subunits of GABA A receptors was observed when both EMC3 and EMC6 were knocked down. We carried out siRNA transfection in HEK293T cells stably expressing α1β2γ2 GABA A receptors; 48 h after transfection, proteins were extracted from cell lysates and incubated with anti-α1 antibody. The immuno-purified eluents were separated through SDS-PAGE gel, and western blot analysis was performed to detect SEC61α and α1 subunits. Quantification of the band intensity of SEC61α over α1 after immunoprecipitation was shown on the right (n = 3). Each data point is presented as mean ± SEM ∗, p< 0.05; ∗∗, p< 0.01. NT: Non-targeting scrambled siRNA; IP: immunoprecipitation; EV: empty vector; WT: wild type.

Article Snippet: The human FLAG-tagged EMC5 plasmid (catalog #: RC207046) and FLAG-tagged EMC6 plasmid (catalog #: RC215548) were obtained from OriGene.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Negative Control, Sequencing, Mutagenesis, Transfection, Stable Transfection, Expressing, Incubation, Magnetic Beads, Purification, SDS Page, Western Blot, Plasmid Preparation

Journal: iScience

Article Title: The endoplasmic reticulum membrane complex promotes proteostasis of GABA A receptors

doi: 10.1016/j.isci.2022.104754

Figure Lengend Snippet: Overexpression of EMC3, and EMC5 and EMC6 restores surface expression and whole-cell currents of disease-associated variants of GABA A receptors (A–C). Overexpression of EMC3 and EMC5/6 increased surface expression of α1 subunits of GABA A R in HEK293T cells stably expressing α1(D219N)β2γ2 (A), α1(G251D)β2γ2 (B) and α1(P260L)β2γ2 (C). We carried out cDNA transfection of EMC3, or co-application of EMC5 and EMC6, in corresponding HEK293T cells; 48 h after transfection, surface proteins were enriched through biotin-neutravidin affinity purification, and western blot analysis was applied to detect α1 subunits. Na + /K + ATPase served as loading control of cell surface proteins. Normalized surface α1 band intensity was shown below the images (n = 3). (D) HEK293T cells stably expressing α1(G251D)β2γ2 GABA A receptors were transfected with empty vector control (CTL) or EMC5 and EMC6 cDNAs. 48 h after transfection, cycloheximide (CHX) (100 μg/mL), a potent protein synthesis inhibitor, was added to cell culture media for the indicated time. Cells were then harvested, and total proteins were subjected to SDS-PAGE and western blot analysis. Quantification of the α1 band intensity was plotted against the incubation time with CHX (n = 3). (E–G) Increased whole-cell patch-clamping currents of GABA A receptors were recorded in HEK293T cells stably expressing α1(D219N)β2γ2 (E), α1(G251D)β2γ2 (F) and α1(P260L)β2γ2 (G). Transfection of cDNA was applied the same way as in (A–C); 48 h after transfection, patch clamping was performed on the cells with the IonFlux Mercury 16 ensemble plates at a holding potential of −60 mV. GABA (100 μM) was applied for 4 s, as indicated by the horizontal bar above the currents. The peak currents (Imax) were acquired and analyzed by Fluxion Data Analyzer (n = 6 - 10). Each data point is presented as mean ± SEM ∗, p< 0.05; ∗∗, p< 0.01. CTL: Empty vector control sample.

Article Snippet: The human FLAG-tagged EMC5 plasmid (catalog #: RC207046) and FLAG-tagged EMC6 plasmid (catalog #: RC215548) were obtained from OriGene.

Techniques: Over Expression, Expressing, Stable Transfection, Transfection, Affinity Purification, Western Blot, Plasmid Preparation, Cell Culture, SDS Page, Incubation

Journal: iScience

Article Title: The endoplasmic reticulum membrane complex promotes proteostasis of GABA A receptors

doi: 10.1016/j.isci.2022.104754

Figure Lengend Snippet:

Article Snippet: The human FLAG-tagged EMC5 plasmid (catalog #: RC207046) and FLAG-tagged EMC6 plasmid (catalog #: RC215548) were obtained from OriGene.

Techniques: Recombinant, Modification, Transfection, Magnetic Beads, Protease Inhibitor, Mutagenesis, Titration, Plasmid Preparation, Software