For experiments combining FUNCAT with immunocytochemistry, cells that had previously undergone the AHA-DBCO click chemistry protocol (described above) were washed and then blocked for 1 hour in 3% bovine serum albumin (BSA) in PBS, pH 7.4, at room temperature before being incubated with 1:200 rabbit anti-synaptophysin (080139; ThermoFisher) or 1:1000 mouse anti-S6 ribosomal protein (2317; Cell Signaling Technology) primary antibodies overnight at 4°C. Coverslips were then washed with PBS and incubated in 1:500 Cy3-conjugated rabbit anti-mouse (for synaptophysin; GE Healthcare, Buckinghamshire, UK) or goat anti-rabbit (for S6; GE Healthcare, Buckinghamshire, UK) secondary antibody for 1 hour at room temperature before being mounted on slides using ProLong Gold Antifade mountant. Image analysis is described in the next section.
Mouse anti s6 ribosomal protein
Manufactured by Cell Signaling Technology
Mouse anti-S6 ribosomal protein is a primary antibody that recognizes the S6 ribosomal protein. The S6 ribosomal protein is a component of the 40S subunit of the eukaryotic ribosome and is involved in protein synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti phospho s6 ribosomal protein ser235 236, by Cell Signaling Technology (3 mentions)
Rabbit anti synaptophysin, by Thermo Fisher Scientific (1 mentions)
Hybridoma bank, by Developmental Studies Hybridoma Bank (1 mentions)
Rabbit anti phospho drp1 ser616, by Cell Signaling Technology (1 mentions)
Rabbit anti mlc2, by Cell Signaling Technology (1 mentions)
Goat anti tfam, by Santa Cruz Biotechnology (1 mentions)
Odyssey fc device, by LI COR (1 mentions)
Mouse anti mtco1, by Abcam (1 mentions)
Mouse anti alpha tubulin, by Developmental Studies Hybridoma Bank (1 mentions)
Immuno blot pvdf membrane, by Bio-Rad (1 mentions)
Rabbit anti phospho erk, by Cell Signaling Technology (1 mentions)
Rabbit anti p70 s6 kinase, by Cell Signaling Technology (1 mentions)
Mouse anti actin, by Merck Group (1 mentions)
4 protocols using mouse anti s6 ribosomal protein
1
Combining FUNCAT and Immunocytochemistry
2
Mitochondrial Dynamics in Emphysema
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Human lung tissues from normal subjects and emphysema patients and mouse lung samples were homogenized in RIPA buffer with 1× Protease Inhibitor Cocktail and 1× PMSF. The lysates were centrifuged at full speed for 15 min at 4°C and analyzed by Western blotting as previously described (Lin et al., 2017 (link)). The primary antibodies used were rabbit anti-TFAM (1: 2000, Proteintech, Cat# 22586-1-AP), mouse anti-S6 ribosomal protein (1:2000, Cell Signaling Technology, Cat# 2317), rabbit anti-phospho-S6 ribosomal protein (Ser235/236) (1:2000, Cell Signaling Technology, Cat# 4856), mouse anti-OPA1 (clone 18) (1:2000, BD Transduction Laboratories, Cat# 612606), mouse anti-DLP1 (1:2000, BD Transduction Laboratories, Cat# 611113), rabbit anti-phospho-DRP1 (Ser616) (1:1000, Cell Signaling Technology, Cat# 3455S), and mouse anti-alpha-tubulin (1:3000, Developmental Studies Hybridoma Bank, Cat# 12G10).
3
Quantification of Mitochondrial Proteins
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Purified lung epithelial cells were lysed in 2x Western blotting buffer (120 mM Tris-Cl pH 6.8, 4% SDS, 20% Glycerol, 200 mM DTT, 0.02% Bromophenol Blue). The lysates were centrifuged at 16,100 x g for 15 min at 4 °C before loading onto SDS-PAGE. The primary antibodies used were rabbit anti-Raptor (1:2000, EMD Millipore Corporation, Cat# 09-217; RRID:AB_1659713), mouse anti-S6 ribosomal protein (1:2000, Cell Signaling Technology, Cat# 2317; RRID:AB_2238583), rabbit anti-phospho-S6 ribosomal protein (Ser235/236) (1:2000, Cell Signaling Technology, Cat# 4856), rabbit anti-MLC2 (1:500; Cell Signaling Technology, Cat#3672; RRID:AB_10692513), goat anti-TFAM (1: 250, Santa Cruz Biotechnology, Cat# sc-23588; RRID:AB_2303230), rabbit anti-COX10 (1:500, Proteintech, Cat# 10611-2-AP; RRID:AB_2084833), mouse anti-MTCO1 (1:500, Abcam, Cat# ab14705; RRID:AB_2084810), and mouse anti-alpha-tubulin (1:3000, Developmental Studies Hybridoma Bank, Cat# 12G10; RRID:AB_1157911). Western blotting images were captured on a LI-COR Odyssey Fc device. The uncropped images are available in the Source Data file.
4
Western Blot Analysis of Protein Expression
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Total protein extract was obtained from LHA using lysis buffer (50 mM Tris-HCl pH=7.5, 1 mM EGTA, 1 mM EDTA, 1% Triton-X100, 1 mM Sodium Orthovanadate, 50 mM Sodium Fluoride, 5 mM Sodium Pyrophosphate, 0.27 M sucrose). We charged 20 g of total protein in 8-10% SDS-PAGE acrylamide gels.
Proteins were transferred to Immuno-Blot PVDF membranes (BIORAD) with 0.2 or 0.45 g pore size. The membranes were subsequently blocked with 3-5% BSA in TBS 0.1% tween (TBS-T) for 1h. Membranes were incubated overnight with primary antibodies in blocking solution at 1:1000 dilution: goat anti-k-OR (Sigma SAB2501442), rabbit anti-phospho-ERK (Cell Signaling #4370), rabbit anti-p70S6Kinase (Cell Signaling #9202), rabbit anti-phospho-S6 ribosomal protein (Ser235/236) (Cell Signaling #2211), mouse anti-S6 ribosomal protein (Cell Signaling #2317) and mouse anti--Actin (Sigma A5316). Afterwards, membranes were washed three times 10 minutes with TBS-T, incubated 1h with secondary antibodies (DAKO) at 1:5000 dilutions, and again washed before protein detection using ECL chemiluminescent western blot substrate (Thermo Scientific). Images were quantified by ImageJ software.
Proteins were transferred to Immuno-Blot PVDF membranes (BIORAD) with 0.2 or 0.45 g pore size. The membranes were subsequently blocked with 3-5% BSA in TBS 0.1% tween (TBS-T) for 1h. Membranes were incubated overnight with primary antibodies in blocking solution at 1:1000 dilution: goat anti-k-OR (Sigma SAB2501442), rabbit anti-phospho-ERK (Cell Signaling #4370), rabbit anti-p70S6Kinase (Cell Signaling #9202), rabbit anti-phospho-S6 ribosomal protein (Ser235/236) (Cell Signaling #2211), mouse anti-S6 ribosomal protein (Cell Signaling #2317) and mouse anti--Actin (Sigma A5316). Afterwards, membranes were washed three times 10 minutes with TBS-T, incubated 1h with secondary antibodies (DAKO) at 1:5000 dilutions, and again washed before protein detection using ECL chemiluminescent western blot substrate (Thermo Scientific). Images were quantified by ImageJ software.
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