The wild-type (R21) and rodA(p)::mRFP::rodA strains (1 × 107 conidia) were grown 50 ml liquid minimal media plus 1 % fructose at 37 °C in a rotatory shaker (180 rpm) for 16 h. After, the mycelia were transferred to SEB media for 24, 72, and 120 h. The collected mycelia and SEB were immediately frozen and the proteins extracted as previously described. Culture supernatants (40 ml) were freeze dried and resuspended in extraction buffer. Protein content was measured using the Bio-Rad protein assay. Total proteins (20 µg) were separated on a Bolt® 4–12 % Bis–Tris Plus Gel and transferred onto a nitrocellulose membrane using the iBlot®2 dry blotting system (Life Technologies). Coomassie and ponceau stained gels revealed protein loading and correct transfer [62 ]. The primary anti-RFP (ABcam: AB65856) was used at a 1:1000 dilution in TBS-T (25 mM Tris, 0.15 M NaCl, 0.05 % Tween-20, pH 7.5) overnight at 4 °C. Primary antibodies were detected using a horseradish peroxidase (HRP)-conjugated second antibody (Kirkegaard and Perry Laboratories) at a 1:2000 dilution in TBS-T plus 5 % skimmed milk powder for 1 h, at room temperature. Chemiluminescence was detected using Super signal West Pico chemiluminescent substrate (Pierce).
Ab65856
Manufactured by Abcam
Sourced in United Kingdom
Ab65856 is a laboratory equipment product. It is a device designed for specific laboratory applications. The core function of this product is to perform a particular task in a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
A31573, by Thermo Fisher Scientific (2 mentions)
A11039, by Thermo Fisher Scientific (2 mentions)
A10037, by Thermo Fisher Scientific (2 mentions)
Tcs sp5 x, by Nikon (2 mentions)
A10262, by Merck Group (2 mentions)
C2 confocal microscope, by Nikon (2 mentions)
T6793, by Abcam (2 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Mab377, by Merck Group (1 mentions)
Af 212 na, by R&D Systems (1 mentions)
Mca275r, by Bio-Rad (1 mentions)
Ab33786, by Abcam (1 mentions)
Ba 2001, by Vector Laboratories (1 mentions)
Ba 9500, by Vector Laboratories (1 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
Gfp trap agarose, by Proteintech (1 mentions)
Ab13970, by Abcam (1 mentions)
Volocity acquisition software, by PerkinElmer (1 mentions)
Ab76541, by Santa Cruz Biotechnology (1 mentions)
6 protocols using ab65856
1
Fungal Growth and Protein Expression Analysis
2
Immunohistochemical Staining Protocol
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The animals were perfused and processed for histological analysis as described previously.9 (link) Immunohistochemistry was performed as described previously.10 (link) The following primary antibodies were used: rabbit anti-pRPS6 (#2211, 1:400, Cell Signaling Technology, Danvers, MA, USA), goat anti-human GDNF (AF-212-NA, 1:1,000, R&D Systems Europe), rabbit anti-DHFR DD (1:20,000, a kind gift from the Wandless lab that has been used previously21 (link)), mouse anti-RFP (ab65856, 1:1,000, Abcam, Cambridge, UK), mouse anti-NeuN (MAB377, 1:1,000, Merck Millipore, Darmstadt, Germany), rat anti-CD11B (MCA275R, 1:1,000, Bio-Rad Laboratories, Solna, Sweden), and mouse anti-CD8 alpha (ab33786, 1:1,000, Abcam, Cambridge, UK). The secondary antibodies used were as follows: biotinylated horse anti-mouse (BA2001, 1:200, Vector Laboratories, Peterborough, UK), biotinylated horse anti-goat (BA9500, 1:200, Vector Labs), and biotinylated goat anti-rabbit-biotin (BA1000, 1:200, Vector Labs).
3
Co-Immunoprecipitation of FgRbp1 and FgU2AF23
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The FgRbp1-GFP and FgU2AF23-RFP fusion constructs were generated as described in our previous section. Both constructs were verified by DNA sequencing and co-transformed into PH-1. Transformants expressing pairs of fusion constructs were confirmed by western blotting. In addition, the transformants bearing a single fusion construct were used as references. For Co-IP assays, total proteins were extracted and incubated with the GFP-Trap Agarose (ChromoTek, Martinsried, Germany) at 4 °C for 6 h with rotation. After six times of washing, proteins eluted from agarose were analyzed by western blot detection with anti-RFP (ab65856, Abcam, Cambridge, UK, 1:10000 dilution) antibody. The input samples were also detected with monoclonal anti-GAPDH antibody described in the previous section as a reference. To detect whether the interaction between FgRbp1 and FgU2AF23 depended on RNA, the cell lysate was treated with 500 μg/ml RNase A at room temperature for 30 min prior to immunoprecipitation56 (link). The experiment was repeated twice.
4
Derivation and Characterization of mESCs
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Joshua Brickmann (Copenhagen) provided the E14 Ju09 HV H2B‐Tomato mESCs (Morgani et al, 2013 ) and CAG H2B‐eGFP ESCs were derived from mice (Hadjantonakis & Papaioannou, 2004 ). These mESCs were maintained in mESC medium containing 2i/LIF/serum. Cells were passaged twice with 2iL on mouse embryonic fibroblasts and inactivated with no growth factor medium. Cells were then treated for 48 h with 2i/LIF/serum or 2i/Jaki/serum on mouse embryonic fibroblasts. Chimeric embryos were generated by morula aggregation. Clusters of 5 to 10 mESCs were aggregated with wild‐type CD1 morulae and cultured in potassium (K) simplex optimized medium (KSOM; Chemicon) under paraffin oil at 37°C and 5% CO2 until the late blastocyst stage (embryonic day 4.5). Blastocyst embryos were subjected to immunofluorescent staining using anti‐Cdx2 (1:600, Abcam ab76541), anti‐Gata4 (1:100, Santa Cruz Biotech sc‐9053), anti‐Sox2 (1:100, R&D Systems AF2018), anti‐GFP (1:400, Abcam ab13970), and anti‐RFP (1:100, Abcam ab65856) antibodies. Imaging was performed using a Quorum WaveFX spinning disk confocal system and Volocity acquisition software (Perkin Elmer). The frequency of cells localizing to extraembryonic—trophectoderm (TE) positions in the blastocyst was counted. The investigators were not blinded to allocation during outcome assessment, and the experiments were not randomized.
5
Imaging Larval Development Using Confocal Microscopy
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Wandering third instar larvae were heat-fixed for 5 seconds at 70ºC, and mounted in one drop of 60% glycerol before being imaged on either a Leica TCS SP5 X or a Nikon C2 confocal microscope. Images are displayed as either single optical sections or z-stack projections. Image analysis was performed with Fiji. In The following primary antibodies were used for immunofluorescence: chicken 𝛼-GFP (1:1000, Invitrogen #A10262), mouse 𝛼-acetylated tubulin (1:1000, Sigma #T6793), mouse 𝛼-RFP (1:1000, Abcam #ab65856), and rabbit α-Wkdpep (1:1000, generously provided by Amin Ghabrial). Secondary antibodies utilized in this study include: goat 𝛼-chicken 488 (1:1000, Life Technologies #A-11039), donkey 𝛼-mouse 568
(1:1000, Life Technologies #A-10037 ), donkey 𝛼-rabbit 647 (1:1000, Life Technologies #A-31573).
(1:1000, Life Technologies #A-10037 ), donkey 𝛼-rabbit 647 (1:1000, Life Technologies #A-31573).
6
Imaging Larval Development Using Confocal Microscopy
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Wandering third instar larvae were heat-fixed for 5 seconds at 70ºC, and mounted in one drop of 60% glycerol before being imaged on either a Leica TCS SP5 X or a Nikon C2 confocal microscope. Images are displayed as either single optical sections or z-stack projections. Image analysis was performed with Fiji. In The following primary antibodies were used for immunofluorescence: chicken 𝛼-GFP (1:1000, Invitrogen #A10262), mouse 𝛼-acetylated tubulin (1:1000, Sigma #T6793), mouse 𝛼-RFP (1:1000, Abcam #ab65856), and rabbit α-Wkdpep (1:1000, generously provided by Amin Ghabrial). Secondary antibodies utilized in this study include: goat 𝛼-chicken 488 (1:1000, Life Technologies #A-11039), donkey 𝛼-mouse 568
(1:1000, Life Technologies #A-10037 ), donkey 𝛼-rabbit 647 (1:1000, Life Technologies #A-31573).
(1:1000, Life Technologies #A-10037 ), donkey 𝛼-rabbit 647 (1:1000, Life Technologies #A-31573).
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