SUPPLEMENTARY METHODS
Mice
C57bl/6 mice were obtained from Jackson laboratory, USA. Mice of either sex (6-8 weeks old,
with mean body weight-25g) were housed individually in conventional cages. Temperature of the
animal rooms was maintained between 20 to 25 °C. Photoperiod of 12hr light and 12hr dark were
maintained as per CPCSEA (Committee for the Purpose of Control and Supervision of
Experiments on Animals) regulations. Ad libitum supply of food and water were given to the
animals. Experimental animals were age and weight matched. All animal were sacrificed with
ether overdose as per IACUC guidelines.
Mucosal sub-compartment isolation
Human- Colonocytes were isolated from endoscopically obtained mucosal biopsies as
described earlier with a few modifications.1 Briefly, the mucosal biopsies were collected into
DMEM media containing 5X antibiotics. It was then washed 3 times with sterile 1X PBS
containing 5X antibiotics. The colonic crypts from mucosal biopsy were isolated by Ca 2+
chelation method. Briefly mucosal bits were suspended in oxygenated Ca 2+ free K-H Ringer
containing 1X Trypsin EDTA (HiMedia), 2% FBS, and DTT (0.1M) in a shaking incubator at
37°C for 20 minutes. Isolated crypts were centrifuged at 1500rpm for 5 minutes and the pellet
obtained was digested with 1mg/ml Collagenase and 1mg/ml Dispase for a period of 10
minutes. The pellet was re-suspended in 80% FBS+DMSO and cryopreserved until further use.
Mouse- Excised colon was washed with oxygenated Ca 2+ free Krebs-Henseleit (K-H) Ringer.
The colon was everted, ligated at one end and then filled with oxygenated Ca 2+ free K-H
Ringer. After ligating the other end the colon was incubated in a solution of Ca
2+
free K-H
Ringer containing 0.1M EDTA + 2% FBS in a shaking incubator at 37°C for 40 minutes. The
isolated crypts were centrifuged at 500g for 5 minutes. Single cell suspension of crypt pellet was
obtained by enzymatically digesting with 1mg/ml Collagenase type 3 (Worthington) and
1mg/ml Dispase (Sigma) in Ca 2+ containing Ringer for a period of 10 minutes at 37°C. Cell
suspensions were filtered with 40 μm nylon mesh and centrifuged at 500g for 5 minutes. The
viability of the cells was measured using Trypan blue. To get the single cell suspension of
lamina propria, colon was cut longitudinally and cut into small bits. The bits were digested with a
digestion buffer containing 1mg/ml Collagenase type 3 (Worthington) and 1mg/ml Dispase
(Sigma) in Ca 2+ containing Ringer for a period of 10 minutes at 37°C in a shaking incubator.
The isolated cells were pelleted at 500g for 5 minutes. The cells were then pooled with the
epithelial cells for further analysis.
Immunoprecipitation
Total cellular protein was extracted from colonocytes and lysed using a lysis buffer (150 mM
NaCl, 5 mM EDTA, 10 mM Tris HCl pH 7.4 and 1% Triton X 100). 250µg of whole cell extracts
were pre-cleared by incubating with Protein A/G agarose beads and 1 µg of rat IgG monoclonal
antibody for 1 hour at 4°C.The pre-cleared whole cell extract was incubated with primary
antibody (CD 24 Rat monoclonal IgG2b) for 1 hour at 4 °C and finally with protein A/G agarose
beads at 4 °C overnight .The immune complexes were washed with RIPA buffer, boiled with
SDS protein dissociation buffer for 3 minutes and then resolved on 12.5% SDS PAGE.
Mass Spectrometry
The protein bands resolved on the 12.5% SDS PAGE gel were subjected to in-gel digestion with
reduction and alkylation.2 The digested peptides were then re-constituted in 15 μL of 2%
Acetonitrile with 0.1% formic acid. 1 μL of the same was injected on to the column and
subjected to 70 minute RPLC gradient followed by acquisition of the data on LTQ-Orbitrap-MS.
The identity of the target proteins were determined using multiple search engines including
MASCOT, Swiss-Prot, TrEMBL and NCBI.
1
2
Seidelin JB, Horn T, Nielsen OH. Simple and efficient method for isolation and cultivation of
endoscopically obtained human colonocytes. Am J Physiol Gastrointest Liver Physiol
2003;285:G1122-8.
Shevchenko A, Tomas H, Havlis J, et al. In-gel digestion for mass spectrometric characterization
of proteins and proteomes. Nat Protoc 2006;1:2856-60.